Arachidonic acid (AA), a compound secreted by Sertoli cells (SC) in a FSH-dependent manner, is able to induce the release of Ca2+from internal stores in round spermatids and pachytene spermatocytes. In this study, the possible site(s) of action of AA in round spermatids, the signalling pathways associated and the intracellular Ca2+stores targeted by AA-induced signalling were pharmacologically characterized by measuring intracellular Ca2+using fluorescent Ca2+probes. Our results suggest that AA acts by interacting with a fatty acid G protein coupled receptor, initiating a G protein signalling cascade that may involve PLA2 and ERK activation, which in turn opens intracellular ryanodine-sensitive channels as well as NAADP-sensitive channels in acidic intracellular Ca2+stores. The results presented here also suggest that AMPK and PKA modulate this AA-induced Ca2+release from intracellular Ca2+stores in round spermatids. We propose that unsaturated free fatty acid lipid signalling in the seminiferous tubule is a novel regulatory component of rat spermatogenesis.