The role of pertussis toxin (PT)-sensitive and -insensitive guanine nucleotide-binding proteins (G proteins) in the stimulation of Ca2+ mobilization by thrombin was investigated in cultured rat aortic smooth muscle cells. Characterization using immunoblotting with specific antisera indicated the presence in isolated membranes of the G alpha i2, G alpha i3, G alpha s, G beta 35, and G beta 36 protein subunits as well as a lower molecular weight species of unknown identity. To assess the importance of G proteins in the coupling of thrombin receptors to Ca2+ mobilization, we investigated the effect of PT on Ca2+ responses using fluorescence spectroscopy and the Ca2+ indicator dye Fura-2. Pretreatment of cells for 2 h with PT (1 microgram/ml), which produced 91.3% ADP-ribosylation of PT-sensitive G proteins, did not affect the magnitude of thrombin-induced release of Ca2+ from internal stores, suggesting that the residual 8.7% of PT-sensitive G proteins, or PT-insensitive mechanisms, was responsible for Ca2+ release. However, after an 18-h pretreatment with PT, which produced ADP-ribosylation of the total complement of PT-sensitive G proteins, the thrombin-induced peak Ca2+ response was inhibited by approximately 72%, suggesting that the major fraction of the Ca2+ response was mediated by a slowly ribosylating component. The delayed effect of the toxin was not caused by down-regulation of the beta-subunit of G proteins because quantitative immunoblots showed that levels of the beta-subunit remained constant throughout the period of PT pretreatment. It was also not caused by a reduction in the size of the thrombin-releasable Ca2+ pool because Ca2+ release induced by agents that release Ca2+ directly from internal stores, 2,5-di-tert-butylhydroquinone or thapsigargin, was not affected. In addition, the delayed effect of PT could not be explained in terms of differences in thrombin-induced [3H]inositol trisphosphate (IP3) formation because the level of inhibition of IP3 formation after a 2-h PT treatment was similar to that present after an 18-h pretreatment. The results indicate that a slowly ribosylating PT-sensitive species is the major G protein pathway that couples thrombin-receptor activation to Ca2+ mobilization. This G protein appears to be involved not in the mechanisms that generate IP3 but rather possibly in coupling at the level of the intracellular Ca2+ store.