Pressure overload is the major stimulus for cardiac hypertrophy. Accumulating evidence suggests an important role for calcium-induced activation of calcineurin in mediating hypertrophic signaling. Hypertrophy is an important risk factor for cardiovascular morbidity and mortality. We therefore employed an in vitro mechanical stretch model of cultured neonatal cardiomyocytes to evaluate proposed mechanisms of calcium-induced calcineurin activation in terms of inhibition of calcineurin activity and hypertrophy. The protein/DNA ratio and ANP gene expression were used as markers for stretch-induced hypertrophy. Stretch increased the calcineurin activity, MCIP1 gene expression and DNA binding of NFATc as well as the protein/DNA ratio and ANP mRNA in a significant manner. The specific inhibitor of calcineurin, cyclosporin A, inhibited the stretch-induced increase in calcineurin activity, MCIP1 gene expression and hypertrophy. The L-type Ca<sup>2+</sup> channel blocker nifedipine and a blocker of the Na<sup>+</sup>/H<sup>+</sup> exchanger (cariporide) both suppressed stretch-dependent enhanced calcineurin activity and hypertrophy. Also application of a blocker of the Na<sup>+</sup>/Ca<sup>2+</sup> exchanger (KB-R7943) was effective in preventing calcineurin activation and increases in the protein/DNA ratio. Inhibition of capacitative Ca<sup>2+</sup> entry with SKF 96365 was also sufficient to abrogate calcineurin activation and hypertrophy. The blocker of stretch-activated ion channels, streptomycin, was without effect on stretch-induced hypertrophy and calcineurin activity. The present work suggests that of the proposed mechanisms for the calcium-induced activation of calcineurin (L-type Ca<sup>2+</sup> channels, capacitative Ca<sup>2+</sup> entry, Na<sup>+</sup>/H<sup>+</sup> exchanger, Na<sup>+</sup>/Ca<sup>2+</sup> exchanger and stretch-activated channels) all but stretch-activated channels are possible targets for the inhibition of hypertrophy.