Objective. According to our recent data, maturation of sympathetic control in rats over the first month of postnatal development is accompanied by a decrease of Ca²⁺-sensitivity of vascular smooth muscle (VSM) contraction, whereas chronic sympathetic denervation reverses these changes. The present study tested the hypothesis that long-term effect of sympathetic nerves on Ca²⁺-sensitivity is ascribed to alteration of Rho-kinase expression or activity. Methods. Experiments were done on newborn (NB: 1–2 wk old), adult control (AD) and adult sympathectomised (SNX) rats. Sympathectomy was provided by injections of guanethidine on postnatal days 1–42. Contraction and [Ca²⁺]_i in saphenous arteries were studied using wire-myography and FURA-2 fluorimetry. Rho-kinase abundance in this artery was estimated by Western blot analysis. Arterial blood pressure (BP) was recorded in urethane-anesthetised (NB vs AD) or in conscious (SNX vs AD) rats. Results. Rho-kinase abundance was 1.5-fold higher in NB compared to AD. Measurements of isometric force vs [Ca²⁺]_i revealed higher Ca2+-sensitivity of contraction in NB than in AD, in response to adrenoceptor agonist methoxamine (MX). Rho-kinase inhibitor Y27632 (3 mM) had little effect on Ca2+-sensitivity in AD, but almost completely inhibited MX- induced Ca²⁺-sensitisation in NB. Under in vivo conditions the pressor response to MX was inhibited by Y27632 (1 mg/kg) or fasudil (3 mg/kg) much stronger in NB than in AD rats. The pattern of vascular alterations in SNX was similar to NB. As compared to AD, SNX showed 1.6-fold higher Rho-kinase abundance and higher effects of fasudil on adrenergic contraction, both in-vitro and in-vivo. Conclusions. Higher Ca²⁺-sensitivity of VSM contraction in NB is attributed to increased Rho-kinase activity. Maturation of sympathetic innervation results in gradual drop of Rho-kinase activity in VSM; the effect may be prevented by chronic denervation.