The present study examines novel mechanisms that regulate levels of the RIa subunit of cAMP-dependent protein kinase. We found that RIa protein is induced threefold by 8-(4-chlorophenyl)thio-cAMP in hormone responsive rat Sertoli cells, while total RIa mRNA is not correspondingly induced. Two RIa mRNA isoforms with different 5 0 untranslated sequences (RIa1a and RIa1b ) are produced from the RIa gene in Sertoli cells. Deletion/mutation analysis of the cAMP-response-element-containing promoter upstream of the RIa exon 1b revealed that while mutation of the cAMP response element had no effects on cAMPmediated induction, a 73-bp region of the RIa exon 1b itself conferred a fivefold to eightfold induction of reporter activity to homologous and heterologous promoters. The responsiveness of this region was dependent on a sense orientation downstream of the promoter start sites and had no effect on reporter mRNA, indicating that the cAMP-mediated induction occurs at the post-transcriptional level. Modeling of the RIa1b 5 0 UTR secondary structure revealed a 5 0 CAPproximal, strong stem-loop presenting an element similar to multiple start-site element downstream-1 (GCTCGG) in the loop region. RNA-EMSAs performed with the labeled RIa1b 5 0 UTR showed stabilization of a protein/RNA complex in extracts from 8-(4-chlorophenyl)thio-cAMP stimulated Sertoli cells. This complex was abolished by mutation of the multiple start-site element downstream-1-like element. Our findings indicate that there is a cAMP-mediated induction of RIa expression at the post-transcriptional level, dependent on the 5 0 UTR of RIa1b mRNA.