We studied the effects of the protein phosphatase (PP) inhibitor cantharidin (Cant) on time parameters and force of contraction (FOC) in isometrically contracting electrically driven guinea pig papillary muscles. We correlated the mechanical parameters of contractility with phosphorylation of the inhibitory subunit of troponin (TnI-P) and with the site-specific phosphorylation of phospholamban (PLB) at serine-16 (PLB-Ser-16) and threonine-17 (PLB-Thr-17). Cant (after 30 min) started to increase FOC (112 ± 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 ± 5 and 128 ± 7% of control) without any alteration of relaxation time. Cant (10 μM) started to increase PLB-Ser-16, but the relaxation was shortened at only 100 μM (from 140 ± 9 to 116 ± 12 ms, n = 9). Moreover, 100 μM Cant, 3 min after application, started to increase PLB-Thr-17, TnI-P, and FOC. Cant (100 μM) began to increase PLB-Ser-16 after 20 min. This was accompanied by shortening of relaxation time. Differences in protein kinase activation or different substrate specificities of PP may explain the difference in Cant-induced site-specific phosphorylation of PLB in isometrically contracting papillary muscles. Moreover, PLB-Thr-17 may be important for inotropy, whereas PLB-Ser-16 could be a major determinant of relaxation time.