In the present study, we investigated the actions of calmodulin (CaM) and CaM-dependent protein kinase II (CaMK-II) on the L-type Ca<sup>2+</sup> currents (I<sub>Ca(L)</sub>) of cultured vascular smooth muscle (VSM) cells (A7r5 cell line), using the whole-cell voltage clamp method. The peak I<sub>Ba</sub> (Ca<sup>2+</sup> channel using 5 m M Ba<sup>2+</sup> as charge carrier) was evoked every 15 s by a test potential to +10 mV from a holding potential of –60 mV. To test the effect of CaM on I<sub>Ba</sub>, 1 µ M calmidazolium (CMZ), an inhibitor of CaM, was added to the pipette solution (pCa of 6.5 or 300 n M [Ca]<sub>i</sub>). The amplitude of maximally activated I<sub>Ba</sub> was –4.3 ± 0.5 pA/pF (n = 13) for control and –8.1 ± 0.9 pA/pF (n = 14) in the presence of CMZ. This difference was statistically significant (p = 0.016). The CMZ stimulation of I<sub>Ba</sub> was not abolished when 5 µ M KN-62, a specific inhibitor of CaMK-II, was included in the pipette (–9.5 ± 1.1 pA/pF; n = 10). Introduction of CaMK-II itself intracellularly had no effect on the basal I<sub>Ba</sub>. On the other hand, the CMZ stimulation of I<sub>Ba</sub> was prevented by both H-7, a nonspecific protein kinase inhibitor, and H-89, a specific inhibitor of protein kinase A (PK-A). Since CMZ is a strong inhibitor of Ca<sup>2+</sup>/CaM-dependent phosphodiesterase (type I PDE), we studied the effect of 8-methoxymethyl-3-isobutyl-1-methylxanthine (MIBMX), another specific inhibitor of the PDE. MIBMX, like CMZ, stimulated I<sub>Ba</sub>: control, –4.6 ± 0.4 pA/pF (n = 10); MIBMX, –9.6 ± 1.2 (n = 8), and CMZ, –7.9 ± 0.9 (n = 15). 0.1 m M 8Br-cAMP, a membrane permeable cAMP analogue, stimulated I<sub>Ba</sub> by +42%: before, –3.7 ± 0.7 pA/pF; after, –5.2 ± 1.0 (n = 6). In conclusion, Ca<sup>2+</sup> channels of VSM cells might not be directly regulated by the CaM/CaMK-II pathway. Therefore, the CMZ stimulation of I<sub>Ba</sub> might occur due to the increase in intracellular concentration of cAMP produced by inhibition of CaM-dependent PDE.