The effect of cAMP on the in vitro expression of rabbit aortic fibronectin was examined using a previously characterized organ culture system. Elevation of intracellular cAMP in incubated aortic rings by use of forskolin or dibutyryl cAMP (dbcAMP) inhibited the normally observed increase in fibronectin mRNA to levels below that found in unincubated tissue. The effect of dbcAMP on fibronectin mRNA was dose dependent and reversible. dbcAMP did not affect overall protein biosynthesis or the changes in collagen or elastin mRNAs that normally occurred during in vitro incubation, suggesting a selective regulatory effect on fibronectin. The inhibitory effect of dbcAMP on steady-state fibronectin mRNA levels was independent of the dibutyrate moiety, was not a result of cytotoxicity, did not require de novo protein synthesis, and did not appear to occur through a protein kinase A pathway. The data suggested that suppression of fibronectin mRNA levels potentially occurred via an indirect mechanism that may have involved a dbcAMP-induced reduction in intracellular calcium ([Ca 2+],) levels. The resultant decrease in [Ca 2+]j may have affected fibronectin expression via a reduction in protein kinase C activity but did not depend on a calmodulin or calmodulin kinase I or II mechanism.