By reverse transcription (RT)-PCR analyses, human gingival fibroblasts (HGFs) were demonstrated to express mRNAs for protease-activated receptor-1 (PAR-1) and PAR-3, although the expression of PAR-3 was much weaker than that of PAR-1. The mRNAs for PAR-2 and PAR-4 were not found by RT-PCR. Furthermore, PAR activation was studied by monitoring cytosolic Ca(2+) concentration ([Ca(2+)]i) in cultured HGFs loaded with fura-2. At concentrations > 0.1 nM, alpha-thrombin caused a transient increase in [Ca(2+)]i in a concentration-dependent manner, and the maximum response was obtained with 10 nM alpha-thrombin. After the [Ca(2+)]i response, the HGFs were completely desensitized to the second stimulation with alpha-thrombin. The PAR-1 agonist peptide SFLLRN produced approximately the same transient [Ca(2+)]i response as alpha-thrombin. After stimulation with SFLLRN, the HGFs did not respond to alpha-thrombin, indicating that treatment with SFLLRN results in complete desensitization to alpha-thrombin. The PAR-2 and PAR-4 agonist peptides had no effect on [Ca(2+)]i in HGFs. These results suggest that alpha-thrombin-induced Ca(2+) mobilization in HGFs is solely mediated by PAR-1.