HIV reverse transcrlptases (RTs) have few cysteine residues rda t.ive to other RTs and retain their DNA polymerization functions followin8 eheJ~),ieal modification by thiol.speeifie reagents. The functional role or ire cysteines in the fidelity of the DNA-dependent DNA synthesis of HIV RTs has been addressed by chemical modification of the wild-type enzy:.~es in combination with the analysis of an ermymatically active mutant HIV-I RT in which all eysteines were modified to serines. We have observed an increase in Y-terminal mispair extension efficiency exhibited by ehemi,:~.'dly modifi~l HIV-I and HIV-2 RTs. The possible involvement of eysteine residues was further substantiated asia8 the cysteine.free mutant I-IlIV-i RT that displays an increased efficiency of mispair extension. These results provide evidence for a possible role of,~steine residues in the flt"~:lity of DNA synthesis catalyzed by HIV RTs,