Calcineurin, a Ca2+-calmodulin-dependent protein phosphatase (PP2B) is one of the links between Ca2+ signals and regulation of gene transcription in cardiac muscle. We studied the Ca2+ signal specificity of calcineurin activation experimentally and with modelling. In the rat atrial preparation, an increase in pacing frequency increased nuclear activity of the calcineurin sensitive transcription factor, Nuclear Factor of Activated T-cells, 2-fold in a cyclosporine- sensitive manner. In line with this, modeling results predicted that the frequency of cardiac Ca2+ transients encodes the stimulus for calcineurin activation. We further observed experimentally that calcineurin inhibition by cyclosporine A modulated Ca2+ release in a Ca2+-dependent manner. Cyclosporine had no effect on [Ca2+]i at pacing frequency of 1 Hz but it significantly suppressed the amplitude of Ca2+ transients, systolic [Ca2+]i and time averaged [Ca2+]i at 6 Hz. Calcineurin had a differential role in the expression of immediate- early genes B-type natriuretic peptide (BNP) and c-fos. Cyclosporine inhibited the pacing-induced BNP gene expression, whereas pacing alone had no effect on the expression of c-fos. However, in the presence of cyclosporine, c-fos mRNA levels were significantly augmented by increased pacing frequency. These results show that frequency-dependent calcineurin activation has a specific role in [Ca2+]i regulation and gene expression, constantly recruited by varying cardiac Ca2+ signals.