1. Gentamicin (10(-4)-10(-2) M) and Co2+ (10(-4)-10(-2) M) produced a decrease in developed tension of canine isolated ventricular muscles leading to near abolition at 10(-2) M. Their negative inotropic effects developed rapidly and wore off shortly after wash-out. 2. The concentration-negative inotropic effect curves for gentamicin were shifted to the right in a parallel manner by increasing external Ca2+, or by the presence of Bay K 8644 (10(-7)-10(-5) M) or isoprenaline (10(-7)-10(-5) M). IC50 values for gentamicin increased about 3-fold with about a 6 fold increase in external Ca2+. The Schild plot yielded a pA2 of 2.29 for Ca2+ and its slope was -1.17 (r = -0.79). 3. The concentration-negative inotropic effect curves for Co2+ were shifted to the right in a parallel manner by increasing external Ca2+, or by the presence of isoprenaline (10(-7)-10(-5) M). IC50 values for Co2+ increased about 5 fold with about a 6 fold increase in external Ca2+. The Schild plot gave a pA2 value of 2.60 for Ca2+ and its slope was -1.11 (r = -0.86). 4. The concentration-positive inotropic effect curves for Ca2+, which were computer-fitted to a logistic equation, gave 2.88 x 10(-3) M for EC50. This value was very close to the KCa calculated from pA2 values for Ca2+ based on antagonism between gentamicin or Co2+ and Ca2+ (5.13 x 10(-3) and 2.51 x 10(-3) M). 5. It is concluded that like Co2+, gentamicin molecules compete with Ca2+ for the same binding sites presumably located at the outer orifice of Ca-channels in the cardiac sarcolemma.