Radiation exposure increases vascular responsiveness and this change involves endothelial damage as well as direct effects on vascular smooth muscle. In this study, we tested the hypothesis that myofilament Ca²⁺ sensitivity in vascular smooth muscle is increased from single whole-body gamma-irradiation (6 Gy). We measured contractile responses from intact and permeabilized rat thoracic aortic rings combined with cytosolic Ca²⁺ ([Ca²⁺]_i) measurements. The sensitivity to KCl and phenylephrine increased significantly in tissues from animals on the 9^th and 30^th days post-irradiation as compared to control. Irradiation also significantly increased Ca²⁺ sensitivity in -escin permeabilized smooth muscle on the 9^th and 30^th days post-irradiation. Inhibitors of protein kinase C, chelerythrine and staurosporine, had no effect on the pCa-tension curves in control permeabilized tissues but significantly decreased Ca²⁺ sensitivity in permeabilized tissues on the 9^th and 30^th days post-irradiation. Phorbol dibutyrate (PDBu, 10^-7 M) increased Ca²⁺ sensitivity in control skinned smooth muscle but was without effect in irradiated vascular rings. Simultaneous measurement of contractile force and [Ca²⁺]_i showed that myofilament Ca²⁺ sensitivity defined as the ratio of force change to [Ca²⁺]_i significantly increased following -irradiation. PDBu (10^-6 M) stimulation of intact aorta produced a sustained contraction while the increase in [Ca²⁺]_i was transient. In irradiated tissues, PDBu-induced contractions were greater than that seen in control tissues but there was no elevation in [Ca²⁺]_i. Taken together, these data strongly support the hypothesis that irradiation increases the sensitivity of vascular smooth muscle myofilaments to Ca²⁺ and this effect is dependent on activation of PKC.