Short-term (less than 2 h) glucose stimulation of isolated pancreatic islets specifically increases the biosynthesis of proinsulin and its converting enzymes PC2 and PC3 at the translation level. To determine whether gene expression of PC2 and PC3 was also regulated by longer-term (more than 6 h) glucose stimulation along with that of preproinsulin, studies were performed with the beta TC3 insulin-producing cell line. By Northern blot analysis, glucose maintained PC2 and PC3 mRNA levels in parallel with those of preproinsulin. After 48 h, mRNA levels of preproinsulin, PC2 and PC3 were, respectively, 2.9 (P < 0.05), 3.0 (P < 0.005) and 5.3 (P < 0.001) times greater in the presence of glucose than in beta TC3 cells cultured in the absence of glucose. Glucose-regulated PC2 and PC3 gene expression, like that of preproinsulin, was maximal at glucose concentrations above 5.5 mM. Studies of mRNA stability showed that the half-lives of PC2 (9 h) and PC3 (5 h) mRNA were much shorter than that of preproinsulin mRNA (over 24 h), but little effect of glucose on stability of these mRNAs was observed. Nuclear run-off analysis indicated that transcription of preproinsulin, PC2 and PC3 was modestly induced after 1 h exposure to 16.7 mM glucose. Therefore preproinsulin, PC2 and PC3 mRNA levels in beta TC3 cells were most probably maintained at the level of gene transcription. In contrast, elevation of cyclic AMP by forskolin had no effect on mRNA levels or gene transcription of preproinsulin, PC2 and PC3, despite a cyclic-AMP-induced phosphorylation of the cyclic AMP response element binding protein that correlated with a marked increase in cJun and cFos gene transcription in the same beta-cells. These results suggest that preproinsulin, PC2 and PC3 gene transcription can be specifically glucose-regulated in a mechanism that is unlikely to involve a key role for cyclic AMP. The co-ordinate increase in PC2 and PC3 mRNA levels with that of preproinsulin mRNA in response to chronic glucose represents a long-term means of catering for an increased demand on proinsulin conversion.