Each segment along arterial vessels adapts to different circumstances, including blood pressure and sympathetic innervation. PKC and Rho-associated kinase (ROCK) Ca(2+)-sensitizing pathways leading to myosin phosphatase inhibition are critically involved in α(1)-adrenoceptor-mediated vascular smooth muscle contraction in distinctive time-dependent manners. We tested whether the amplitude and time course of each pathway varies dynamically between arterial segments. Using pharmacological approaches, we determined the time-dependent roles of Ca(2+) release, Ca(2+) influx, PKC and ROCK in α(1)-agonist-induced contraction and phosphorylation of key proteins in denuded rat small mesenteric artery, midsized caudal artery and thoracic aorta. SR Ca(2+) release and voltage-dependent Ca(2+) influx were essential for the initial rising and late sustained phases, respectively, of phenylephrine-induced contraction, regardless of arterial size. In small mesenteric arteries, α(1A)-subtype-specific antagonists and inhibitors of PKC, but not ROCK, markedly reduced the initial and late phases of contraction in a non-additive manner and suppressed phosphorylation of myosin light chain (MLC) and CPI-17, but not myosin targeting subunit of myosin light chain phosphatase (MYPT1). In aorta, an α(1D)-specific antagonist reduced both the initial and late phases of contraction with a significant decrease in MLC but not CPI-17 or MYPT1 phosphorylation. ROCK inhibitors, but not PKC inhibitors, suppressed the sustained phase of contraction with a decrease in MLC and MYPT1 phosphorylation in the aorta. The effect of ROCK inhibitors was additive with the α(1D)-antagonist. The results for midsized arteries were intermediate. Thus, the PKC-CPI-17 Ca(2+)-sensitizing pathway, which is dependent on PKC subtype and a Ca(2+)-handling mechanism, and is downstream of α(1A) receptors, plays a major role in α(1)-agonist-induced contraction of small resistance arteries in the splanchnic vascular beds. The effect of PKC and ROCK increases and decreases, respectively, with decreasing arterial size.