This study examined the involvement of cyclic GMP, protein kinase G and intracellular Ca2+ movements in the modulation of aqueous humour formation. Using the bovine arterially-perfused eye preparation, drug effects on intraocular pressure and aqueous humour formation rate were measured by manometry and fluorescein dilution, respectively. Drug effects on intracellular [Ca2+] were determined by fura-2 fluorescence ratio technique in nontransformed, cultured ciliary epithelium. Intra-arterial injection of atriopeptin (50 pmol) or sodium azide (10 nmol) produced significant reduction in aqueous humour formation (>38%). This was blocked by selective inhibition (KT-5823) of protein kinase G, but not by selective inhibition (KT-5720) of protein kinase A. Reductions of intraocular pressure produced by atriopeptin or azide were almost completely blocked by KT-5823. ATP (100 microM) caused rapid, transient increase in intracellular Ca2+ followed by a slow decline and prolonged plateau. This response showed concentration-dependent inhibition by atriopeptin, azide or 8-bromo cyclic GMP, and this inhibition of the rapid (peak) Ca2+ increase was enhanced by zaprinast (100 microM; phosphodiesterase inhibitor). KT-5823 blocked the suppression of the peak Ca2+ response but not suppression of the plateau. Arterial perfusion of ATP (0.1-100 microM) produced a concentration-dependent decrease in aqueous humour formation. Aqueous humour formation in the bovine eye can be manipulated through cyclic GMP, operating via protein kinase G. Close parallels appear when Ca2+ movements are modified by similar manipulations of cyclic GMP, suggesting that Ca2+ transients may play an important role in aqueous humour formation and that interplay occurs between cyclic GMP and Ca2+.