To determine the characteristics of cardiac myosin in the conduction system, a pure Purkinje fiber preparation, consisting of atrioventricular nodes and the ventricular conduction system, was obtained from bovine hearts. Two types of myosin heavy chain isozymes, alpha-type and beta-type, were fractionated by affinity chromotography using monoclonal antibodies CMA19 and HMC50, which are specific for the alpha-type heavy chain and beta-type heavy chain, respectively. Competitive enzyme-linked immunosorbent assay demonstrated that the content of beta-type in the atrioventricular node (30-40%) was higher than that in atrial ordinary myocardium (10-20%) and that of the alpha-type was 30-40% in the ventricular conduction system, which was much higher than that in the ventricular ordinary myocardium (less than 10%). By one- and two-dimensional electrophoresis of the peptides produced by partial and complete digestion, the peptide compositions of alpha-type and beta-type in the conduction system were shown to be very similar to those of alpha-type and beta-type in ordinary myocardium, respectively. The CA2+-activated ATPase activity of myosin of the atrioventricular nodes was lower than that of ordinary atrial myosin (0.46 +/- 0.03 versus 0.58 +/- 0.02 mumol Pi/mg/min, mean +/- SEM, p less than 0.05) and in contrast, that of ventricular specialized myocardium was higher than that of myosin in the ventricular ordinary working myocardium (0.32 +/- 0.03 versus 0.22 +/- 0.01 mumol Pi/mg/min, p less than 0.05). This was in good agreement with the relative proportion of myosin isozymes.(ABSTRACT TRUNCATED AT 250 WORDS)