It is well established that LH action is mediated primarily by adenylate cyclase/cAMP. However, the role of inositol phosphate/calcium in LH signaling is less well established. We examined the effects of gonadotropins in primary culture human granulosa-lutein cells and in HEK293 cells transiently transfected with human wild-type or chimeric gonadotropin receptors. The intracellular free calcium concentration was measured using fura-2 microspectrofluorometric techniques. Human (h) LH (2-4 microg/ml) and CG (10 IU/ml) consistently evoked oscillatory calcium signals in HEK293 cells transfected with hLH receptor, whereas hFSH (2-4 microg/ml) failed to elicit any response. Conversely, both hLH and hFSH failed to elicit a calcium response from HEK293 cells transfected with hFSHR, indicating the specificity of the response to the LH receptor. Pretreatment of transfected HEK293 cells with pertussis toxin (100 ng/ml) attenuated all gonadotropin-evoked calcium mobilization. Studies with chimeric LH receptor showed that the sequence of the long extracellular portion of the receptor was not critical for stimulation of PLC activity, but maintained agonist binding specificity. The C-terminal sequence of the receptor was clearly important for the generation of the basal calcium oscillations, but the precise extent of the critical sequence has yet to be identified. Although various subdivisions of this region were capable of stimulating calcium transients, an intact carboxyl-terminal third of the receptor was required for normal and sustained intracellular calcium signaling. Our study unequivocally shows that the hLH receptor is coupled to the inositol phosphate/calcium signaling pathway via a pertussis toxin-sensitive G protein-coupled receptor.