Recently we have shown that chronic exposure of cardiomyocytes to a-adrenoceptor stimulation maintains normal, load-free contraction in hypertrophic cardiomyocytes by upregulation of SERCA via calcium/NFAT signalling. In this study we analysed, if b-adrenoceptor stimulation, which has no growth promoting effects on freshly isolated cardiomyocytes of rat, can also modify cardiomyocyte contraction. As analysed in retardation assays, stimulation of cardiomyocytes with 1mM Iso increased NFAT binding activity within 2 h. SERCA protein expression was also increased by Iso. This SERCA induction was inhibited by NFAT-decoy oligos. Cardiomyocytes, exposed to Iso for 24 h, displayed enhanced relaxation velocity when stimulated at 0.5, 1 or 2 Hz under load free conditions. Decreased cell shortening was found in cardiomyocytes exposed to Iso for 24 h when stimulated at 0.5 Hz, but not at high beating frequencies. In cardiomyocytes, transformed with NFAT-decoy oligos, decreased cell shortening due to 24 h exposure to Iso was also observed under high beating frequencies. Transformation of cardiomyocytes with a mutant oligo, that was unable to block NFAT activity in the cells, did not reduce cell shortening under stimulation with Iso at 2.0 Hz. In conclusion, a functional deficit in contraction of cardiomyocytes due to Iso stimulation can be antagonized by enhancement of relaxation velocity via Iso-dependent and NFAT-mediated activation of SERCA expression.