The purpose of this investigation was to study the effectiveness of two nickel-binding amino acids, histidine (His) and cysteine (Cys), to prevent the inhibitory action of Ni2+ on testosterone (T) production by mouse primary Leydig cell culture. The maximal human chorionic gonadotropin (hCG)-stimulated T response was measured by radioimmunoassay (RIA) in the culture media. Three types of experiments were performed. In a concentration-response study, Ni2+ (62.5 to 1,000 microM) was added to the cells simultaneously with equimolar or twice the equimolar concentrations of His or Cys and the cultures were maintained for 48 h. Nickel-induced reduction in T production was completely prevented by equimolar concentrations of His at Ni2+ concentrations of 125, 250, and 500 microM; equimolar or twice the equimolar concentrations of His were only partially effective at 1,000 microM Ni2+. Protective action of Cys was complete only at the lowest concentration of Ni2+ (125 microM). In a second series, the cells were incubated for various times (0.5 to 48 h) with 1,000 microM Ni2+ in the presence of 2,000 microM His or Cys. Increasing the time of incubation, the protective effect of both amino acids against Ni2+ was reduced. In a third series, attempts were made to reverse the action of 1,000 microM Ni2+ after incubation with cells for various times (0.5 to 24 h), followed by exposure to 2,000 microM His or Cys. Cell cultures were maintained for 48 h. A partial recovery of hCG-stimulated T production could be observed only if the amino acid was added not later than 4 h after the metal. This time was also required to elicit the T depression produced by Ni2+. Administration of either His or Cys at later times had no effect. Our results show that both His and Cys are able to moderate the effects of Ni2+ on Leydig cell T production, depending on the concentration of this metal ion, as well as on amino acid. However, at higher Ni2+ concentrations the complete protection by His or Cys is only temporary. Administration of these amino acids after the Ni2+-produced decrease in T production was not able to reverse the process.