Attenuation of the adrenocortical response to ACTH in septic shock is a well recognised phenomenon, although the underlying mechanisms remain poorly understood. The evidence points towards factors in septic serum that dampen the adrenocortical responsiveness. The aim of this work is to investigate the cellular function of adrenocortical cells in septic shock and to determine the components within serum that could influence their function. Septic rat serum was collected from male Sprague-Dawley rats 24 or 48 hr after induction of sepsis by intraperitoneal injection of faecal slurry. Healthy primary rat adrenal cell cultures were incubated with septic serum and stimulated with ACTH or forskolin for 60 min prior to harvesting of cells and medium for cAMP assay. Septic serum both shifted the EC50 from 0.20×10−9 M (control) to 1.20×10−9 M (septic) and reduced the maximum effect by 60%. Furthermore, the production of cAMP was impaired by 55% (P=0.012, n=7) when stimulated by forskolin (10−5 M) in the presence of septic serum. This suggests that septic serum impairs the function of adenylate cyclases in the generation of cAMP. There are at least nine adenylate cyclases (AC) known. Using primers designed to all nine AC, we demonstrated that 4 AC are present in rat adrenals in vivo AC 3, 4, 6 and 9. Expression of AC 3 and 6 was attenuated in septic adrenals. Further quantification was performed using real time RT-PCR which confirmed that in sepsis AC 3 expression is impaired by 47% (P=0.015, n=3) and AC 6 expression is impaired by 70% (P=0.0014, n=4). These findings suggest that septic serum impairs the function of adrenocortical cells in part via impaired AC3 and AC6 gene expression.