A quantitative relationship between rat liver mitochondrial matrix free Ca2+ ([Ca2+]m) and citrullinogenesis has been observed. Maximum citrulline synthesis occurred at 100 to 200nM [Ca2+]m; higher [Ca2+]m caused inhibition. When [Ca2+]m was decreased to below 50nM, by addition of A23187 and EGTA, inhibition also occurred. By incubating mitochondria with ruthenium red ([Ca2+]m = 200nM) prior to addition of extramitochondrial free Ca2+ ([Ca2+]o) it was found that high external Ca2+ (800nM) did not inhibit citrulline synthesis thus demonstrating that [Ca2+]m, not [Ca2+]o was controlling citrullinogenesis.