The degree of saturation of cytochrome P-450scc with cholesterol and the substrate turnover number of the cytochrome in cultured trophoblasts and mitochondria from the human placenta were investigated. Cholesterol sulfate was found to be a suitable substrate for probing the degree of saturation of cytochrome P-450scc with substrate during culture and in isolated mitochondria, since it enabled the maximum velocity of the cholesterol side-chain cleavage reaction to be estimated. In contrast, 25-hydroxycholesterol and low density lipoprotein supported trophoblast progesterone production at lower rates than that measured with saturating cholesterol sulfate. In the absence of exogenous substrate, the highest rate of progesterone synthesis by trophoblasts was observed at the beginning of the culture. With cholesterol sulfate as substrate, the turnover number of cytochrome P-450scc in cultured cells was 2.8 min-1 and was not significantly different to the turnover number of the cytochrome for placental mitochondria, where cholesterol is known to be saturating. Results indicate that cholesterol is limiting for progesterone synthesis in cultured trophoblasts even in the presence of lipoprotein rich medium and 8-bromo-cAMP. The concentration of cytochrome P-450scc in trophoblasts was only 20% of that measured for placental homogenate suggesting an induction of the cytochrome occurs when the trophoblasts fuse in vivo to form syncytiotrophoblasts.