We previously showed that serotonin (5-Hydroxytryptamine, 5-HT) receptor antagonist methysergide partially inhibited cyclopiazonic acid (CPA)-potentiated and 5-HT-induced contractions suggesting the contribution of protein kinase C (PKC)-mediated receptor internalization in attenuated antagonism. In the present study, the effects of CPA and 5-HT receptor antagonist methysergide and ketanserin on 5-HT-induced calcium responses following PKC inhibition were further investigated. For this purpose, intracellular calcium levels were monitored in A7r5 vascular smooth muscle cells by spectrofluorometry using the fluorescent indicator fura-2. Cells were pre-incubated with specific PKC inhibitor D-sphingosine. In experimental aspects, spontaneous calcium oscillations hindering the monitoring of responses were determined in over confluent cells. 5-HT-induced calcium levels were significantly decreased in D-sphingosine-treated cells compared to control. CPA significantly potentiated 5-HT-induced calcium responses while ketanserin partially but insignificantly inhibited the potentiated responses in the presence of PKC inhibition. In conclusion, increased cell confluency may result in the generation of spontaneous calcium spikes indicating the importance of using optimum cell density for monitoring of intracellular calcium levels. The data suggests that CPA-mediated potentiation and diminished antagonistic effects on 5-HT-induced calcium elevations in vascular smooth muscle cells, are partially mediated by PKC.