Two short-lived precursor proteins, pp37 and pp32, of the mitochondrial phosphoprotein pp30 (formerly denoted as ib) have been detected in Bt2cAMP-stimulated rat adrenal cortex cells, incubated at 25 degrees C or with 1,10-ortho-phenanthroline at 37 degrees C. Subsequently, these two precursor proteins were also identified in cells incubated at 37 degrees C, where they are present only at low levels due to their short half-life. pp30 is produced in several steroidogenic tissues in response to trophic hormone or second messenger analogue. pp37 and pp32 are also phosphoproteins located in the mitochondrion that are produced in response to cAMP analogue and give rise to proteolytic peptide maps similar to that of pp30. As for pp30, inhibition of cytosolic translation prevents the production of pp37 and pp32. The larger precursor protein pp37 has an apparent molecular mass of 37 kDa, an isoelectric point of approximately 7.1, and a half-life at 37 degrees C of 3-4 min. Pulse-chase studies indicate that this protein is processed into the smaller protein, pp32, which has an apparent molecular mass of 32 kDa, an isoelectric point of approximately 6.4, and a half-life at 37 degrees C of 3-4 min. This latter protein is the immediate precursor of pp30. Since ortho-phenanthroline inhibits the mitochondrial processing protease, while the lower incubation temperature slows both protein import and protease processing, the experimental conditions necessary to detect these proteins are consistent with pp37 being a precursor protein that contains two cleavable presequences and is imported into the mitochondrion. The sequential removal of these sequences produces the mature protein pp30.