In previous studies we described the synthesis of three mitochondrial proteins (A, B and C) in response to acute in vitro stimulation by lutropin of small bovine luteal cells. Protein A had a molecular weight of 28 kDa and an isoelectric point (pI) of 6.7. Proteins B and C had a molecular mass of 27 kDa and pI of 6.2 and 6.4, respectively. The appearance of these proteins was prevented by 100 microM cycloheximide. In the present study, we have shown that the time course of synthesis of protein A and its hCG dose-response closely parallel the increase in progesterone production. The induction by hCG of protein A was already observed after a 5 min incubation. Pulse chase experiments by addition of excess unlabelled methionine after prelabelling with [35S]methionine indicated that its half-life was approximately 15-20 min. Study of 32P labelled phosphate incorporation into individual proteins and treatment by alkaline phosphatase of [35S]methionine-labelled proteins demonstrated that none of the three proteins A, B or C was a phosphoprotein. Localization of protein A in mitochondria, at the site of the rate limiting step in steroidogenesis, and the high degree of correlation between its 35S labelling and progesterone production argue in favour of its involvement in the acute regulation of steroidogenesis.