To assess functional and cellular effects of myocardial β1-adrenoceptor overexpression, alterations of the β-adrenergic signal transduction pathway and contractile function in transgenic mice with atrial overexpression of the human β1-adrenoceptor were investigated. Radioligand binding experiments confirmed a 5- to 6-fold increase in β-adrenoceptor density and a 2.7-fold increase in high-affinity binding sites in atria of transgenic mice. Dose–response curves for isoprenaline-induced force of contraction showed unchanged maximum effects but significantly increased pD2values. Basal, MnCl2- and isoprenaline-stimulated adenylyl cyclase activities did not significantly differ, whereas the Gpp(NH)p and forskolin effect tends to be reduced in transgenic mice. The level of Giα (pertussis toxin-catalyzed ADP-ribosylation) was unchanged, whereas the bioactivity of Gsα (reconstitution experiments into S49 cyc−cell membranes) was reduced by about 19% in the transgenic group. These results suggest that overexpressed β1-adrenoceptors act as functional spare receptors. In addition, increased β1-adrenoceptor density is associated with a decrease in Gsα-activity.