In guinea-pig papillary muscles the positive inotropic effect of flosequinoxan (BTS) starting at 100 μmol/1 amounted to 287.6 ± 34.2% at 300 μmol/l without any effects on time to peak tension (103.9 ± 2%) and relaxation time (107.1 ± 6.7% of predrug value, respectively). 10 μmol/l carbachol attenuated the positive inotropic effect of 300 μmol/l to 166.5 ± 11.6% (n = 10). The phosphorylation state of the inhibitory subunit of troponin (TnI) and phospholamban(PLB) in [32P]-labeled guinea-pig ventricular myocytes was increased starting at 100 μmol/l amounting to 142.5 ± 12.6% and 130.9 ± 2.2% at 300 μmol/l, respectively (n = 5). Furthermore, BTS (300 μmol/l) decreased phosphorylase phosphatase activity by 23.1%. It is concluded that the contractile effects of BTS are accompanied by enhanced phosphorylation of regulatory proteins which could in part be due to inhibition of phosphorylase phosphatase activity.