LH treatment of isolated medium (3- to 7-mm) follicles from porcine ovaries stimulated pregnenolone synthesis by mitochondria isolated from these tissues. This LH stimulation of pregnenolone synthesis was observed regardless of whether exogenous cholesterol was added to the mitochondrial assay system, suggesting that factors besides cholesterol availability may be important for mitochondrial steroidogenesis. Since ovarian mitochondria contain AMP-dependent protein kinase activity, we investigated the role of protein phosphorylation in regulating mitochondrial steroidogenesis. Mitochondria were isolated from different sized follicles and corpora lutea and incubated for 10 min with [gamma-32P]ATP in the absence or presence of 2 microM cAMP. Mitochondrial proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 32P labeling of mitochondrial proteins was determined by autoradiography. In all mitochondrial preparations from the various ovarian tissues, 32P labeling was found only for proteins with apparent mol wt of 44,000 and 55,000. While cAMP addition was necessary for phosphorylation of the 44,000 mol wt mitochondrial protein(s), phosphorylation of the 55,000 mol wt mitochondrial protein(s) occurred independently of cAMP. Intact medium follicles were preincubated for 120 min in medium 199D containing 32Pi, and then incubated in nonradioactive medium without or with LH (1 microgram/ml) for up to 2 h. Mitochondria were isolated from these follicles, and their proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. While several mitochondrial proteins were labeled in these experiments, only the 44,000 and 55,000 mol wt proteins were studied. After 30 min of incubation, LH treatment stimulated the phosphorylation of a mitochondrial protein with apparent mol wt of 44,000. However, 32P labeling of this 44,000 mol wt mitochondrial protein decreased in LH-treated follicles after incubation for 60 and 120 min. In untreated follicles, 32P labeling of the 44,000 mol wt mitochondrial protein did not change after 30 min of incubation, but phosphorylation did increase after 60- and 120-min incubations. 32P labeling of the 55,000 mol wt mitochondrial protein increased linearly with time in both untreated and LH-treated follicles. Although cAMP treatment of isolated follicular mitochondria was similar to the LH stimulation of intact follicles with respect to phosphorylation of the 44,000 mol wt mitochondrial protein, cAMP treatment of isolated follicular mitochondria did not stimulate pregnenolone synthesis.(ABSTRACT TRUNCATED AT 400 WORDS)