Relatively little is known about the contribution of Ca²⁺ dependent and independent mechanisms in the contractility of neonatal gastrointestinal smooth muscle. We have therefore studied Ca²⁺ homeostasis and Ca²⁺ sensitization mechanisms in 10-day and adult guinea pig gallbladder smooth muscle to elucidate developmental changes in these processes. Gallbladder contractility was evaluated by isometrical tension recordings from strips, [Ca²⁺]_i was estimated by epifluorescence microscopy of fura-2 loaded isolated cells and protein expression and phosphorylation was assessed by western blot. The neonatal gallbladder contracted significantly less to cholecystokinin (CCK) than adult tissue but this correlated with an increased Ca²⁺ mobilization, suggesting immaturity of Ca²⁺ sensitization mechanisms. The enhanced Ca²⁺ release in newborn gallbladder was the result of the increase in the size of the releasable Ca²⁺ pool. Moreover, in neonatal smooth muscle cells neither the PMCA pump nor the Na⁺/Ca²⁺ exchanger collaborate in the extrusion of Ca²⁺. By the contrary, in these cells there is an increase in phospholamban phosphorylation which could drive to an over-activity of the SERCA pump. The reduced Ca²⁺ sensitivity in neonatal tissues was demonstrated by the lack of effects Y27362, an inhibitor of Rho kinase (ROCK) and GF109203X, an inhibitor of protein kinase C (PKC), on agonist-induced contraction. In addition, neonatal gallbladder showed lower levels of RhoA, ROCK, PKC and the two effectors CPI-17 and MYPT1, and the absence of CPI-17 and MYPT1 phosphorylation in response to agonists. In conclusion, our results indicate that the main mechanisms involved in smooth muscle contractility are under developmental regulation