Stemphylium loti, a pathogen of a cyanogenic plant, possesses a cyanide-insensitive alternate respiratory pathway. In the absence of cytochrome inhibitors, the alternate system had only a minor role in respiration. When S. loti was grown in medium amended with antimycin to block the cytochrome chain, the alternate system accounted for the total oxygen consumption associated with respiration.The contribution of the alternate respiratory system to increases in formamide hydro-lyase (FHL) activity and to growth in S. loti in vitro was assessed. FHL, induced by cyanide, converts cyanide to nontoxic formamide and is partially responsible for the tolerance of S. loti to high concentrations of cyanide in vitro. When the cytochromes were blocked and the cyanide-insensitive respiratory pathway accounted for 100% of the oxygen uptake associated with respiration, FHL activity, but not changes in dry weight, was positively correlated with activity of the alternate pathway. As the alternate pathway activity decreased with increasing concentrations of salicylhydroxamic acid, the level of FHL activity correspondingly decreased. The alternate respiratory system may provide for increases in FHL activity but not for growth. S. loti appears to have two mechanisms for cyanide tolerance in vitro: cyanide-insensitive respiration and FHL activity. The initial activity of FHL for detoxification of cyanide may depend on the alternate respiratory pathway when the cytochromes of the electron transport chain are blocked.