Ca2+ sensitization has been postulated to contribute to the myogenic contraction of resistance arteries evoked by elevation of transmural pressure. However, the biochemical evidence of pressure-induced increases in phosphorylated myosin light chain phosphatase (MLCP) targeting subunit 1 (MYPT1) and/or 17 kDa protein kinase C (PKC)-potentiated protein phosphatase 1 inhibitor protein (CPI-17) required to sustain this view is not currently available. Here, we determined whether Ca2+ sensitization pathways involving Rho kinase (ROK)- and PKC-dependent phosphorylation of MYPT1 and CPI-17, respectively, contribute to the myogenic response of rat middle cerebral arteries. ROK inhibitors (Y27632, 0.03-10 μmol l−1; H1152, 0.001-0.3 μmol l−1) and PKC inhibitors (GF109203X, 3 μmol l−1; Gö6976; 10 μmol l−1) suppressed myogenic vasoconstriction between 40 and 120 mmHg. An improved, highly sensitive 3-step Western blot method was developed for detection and quantification of MYPT1 and CPI-17 phosphorylation. Increasing pressure from 10 to 60 or 100 mmHg significantly increased phosphorylation of MYPT1 at threonine-855 (T855) and myosin light chain (LC20). Phosphorylation of MYPT1 at threonine-697 (T697) and CPI-17 were not affected by pressure. Pressure-evoked elevations in MYPT1-T855 and LC20 phosphorylation were reduced by H1152, but MYPT1-T697 phosphorylation was unaffected. Inhibition of PKC with GF109203X did not affect MYPT1 or LC20 phosphorylation at 100 mmHg. Our findings provide the first direct, biochemical evidence that a Ca2+ sensitization pathway involving ROK-dependent phosphorylation of MYPT1 at T855 (but not T697) and subsequent augmentation of LC20 phosphorylation contributes to myogenic control of arterial diameter in the cerebral vasculature. In contrast, suppression of the myogenic response by PKC inhibitors cannot be attributed to block of Ca2+ sensitization mediated by CPI-17 or MYPT1 phosphorylation.