Various types of protein kinase A (PKA) alterations have been observed in adrenocortical tumours and Carney complex (CNC). PKA is a heterotetramer of two regulatory and two catalytic subunits. The R1A and R2B proteins are the most abundant regulatory subunits in endocrine tissues. A decrease in R2B protein levels has been observed in adrenal adenoma, whereas tumours from patients with CNC display a decrease in R1A protein levels. Dysregulation of the balance between R1A and R2B may thus be involved in adrenal tumourigenesis. We investigated the impact of the differences in the balance of PKA subunits on cell growth using specific cAMP analogues. We assessed the effects of 8-chloroadenosine-cAMP (8Cl-cAMP), a site-selective activator of PKA R2B, in H295R adrenocortical cells. 8Cl-cAMP stimulated PKA activity, decreased R1A levels and increased R2B levels. It had no cytotoxic effects, initially stimulating DNA synthesis and then inducing apoptosis by disrupting G₂/M progression. We observed an initial accumulation of cells in the S phase, translocation of cyclin A to the nucleus, CDK2 activation, sustained DNA synthesis and proliferating cell nuclear antigen accumulation. Cell cycle arrest in the G₂ phase was parallel with the accumulation of cyclin B and the inactivation of CDC2 kinase. The 8CPT-cAMP, which activates the R2B subunit, had similar effects. R2B silencing reduced the apoptosis induced by tumour necrosis factor α and transforming growth factor β. Thus, R2B is a key regulator of proliferation/differentiation in H295R cell line along with the complex balance between the PKA subunits. Activation of PKA R2B and dysregulation of the R1A/R2B balance regulate cell cycle progression and apoptosis in adrenocortical cells by modulating cyclin production and cyclin-dependent kinase activities.