Metallothioneins are forming a superfamily of non-enzymatic polypeptides (61-68 aa), which are distinguished by low molecular weight (6-7 kDa), specific aa combination (containing high amount of cysteine) and high sulfur and metal content. Cysteine ends of metallothioneins are involved in disulfide bridges and also in metal ions. In this study, the goal is to evaluate the binding ability of 4 different isoforms of rice ( Oryza sativa ) metallothionein, to arsenic, chromium and mercury. By now, the gene coding 4 rice metallothioneins isoforms, namely OsMTI-1b, OsMTI-2b, OsMTI-3a and OsMTII-1a, which based on cysteine arrangement at their amino and, belong to types I, II, III and IV, respectively, have been produced heterologously in E.coli bacteria and along with adjoining partner; however, binding ability of these isoforms have been evaluated just in confrontation to divalent metals. In this study, in order to evaluate the binding ability of different Metallothioneins to Cr 3+ , Cr 6+ and As 3+ (as 3- and 6-valent metals) metals and finally Hg 2+ , which has high importance in being toxic, we planned tests in two inter- and intra-cellular level. In intra-cellular level, we drew the growth curve for strains, expressing recombinant proteins, and control strain, in existence of desired metals, and decrease level of these metals from the cell culture, were measured by Atomic Absorption Spectrophotometer. Results showed that expression of recombinant proteins within the expressing strains, caused increase in relative resistance, but differently, and of course more decrease of metals from cell culture media, comparing to control strains, which, in general view, showed the impact of strains expressing various metallothionein isoforms in this level of experiment in the form of R-MTI?R-MTII?R-MTIII?R-MTIV when exposed to Hg 2+ , in the form ofR-MTI?R-MTIII?R-MTII?R-MTIV andR-MTI?R-MTIII?R-MTII?R-MTIV, when exposed to Cr 3+ and Cr 6+ , respectively, and finally in the form of R-MTI?R-MTII?R-MTIII?R-MTIV when exposed to As 3+ in the cell culture media. In order to identify the binding ability of these isoforms to mentioned metals in vitro, first high amount of recombinant protein was produced in cell culture containing ZnSO 4 metal and was purified by using two stages of affinity chromatography and gel filtration. In order to produce Apo protein and in fact omit the ZnSO 4 metal, the method of making the culture acidic was used; the isolated metals were omitted by gel filtration and then protein was exposed high amount of As 3+ , Cr 3+ , Cr 6+ and Hg 2+ and again in order to omit the unbounded metals, gel filtration was done. Besides evaluation of change in absorbance pattern, the binding strength of these metals were evaluated by a competitive reaction with Elman reagent. Results showed that first, in vitro the possibility of binding of metal to metallothioneins exist, however metals bind to various isoforms with different binding strength. So that in this step of experiment this binding strength was identified for recombinant MTI protein as Hg 2+ As 3+ Cr 3+ Cr 6+ , for MTII as Hg 2+ Cr 3+ As 3+ Cr 6+ , for MTIII as Hg 2+ Cr 3+ Cr 6+ As 3+ and finally for MTIV as Hg 2+ Cr 3+ Cr 6+ As 3+ . Keywords : Metallothionein, Protein Expression, Growth Curve, Affinity Chromatography, Gel Filtration, Elman Reagent