We investigated the expression of α₁ and β subunits of the L-type Ca²⁺ channel on the protein level in cardiac preparations from normal human heart ventricles and from the hypertrophied septum of patients with hypertrophic obstructive cardiomyopathy (HOCM). 1,4-Dihydropyridine (DHP) binding and immunorecognition by polyclonal antibodies directed against the C-terminal amino acid sequences of the β₂ and β₃ subunits were used for detection and quantification of α₁, β₂, and β₃ subunits. B_max of high-affinity DHP binding was 35±2 fmol/mg protein in HOCM and 20±2 fmol/mg protein in normal human hearts (P<0.05). In rabbit hearts the anti-β₂ subunit antibody immunoprecipitated 80% of the total amount of DHP-labeled Ca²⁺ channels present in the assay. Under identical experimental conditions 25% of labeled Ca²⁺ channels were recovered in the immunoprecipitates of both normal and HOCM ventricles. A similar partial immunoprecipitation was observed in pig hearts. Immunoblot analysis demonstrated that the β₂ subunit was associated with the DHP receptor/Ca²⁺ channel in cardiac muscle of rabbit, pig, and human heart. In neither of these purified cardiac Ca²⁺ channels was the β₃ subunit isoform detected. Our results suggest that both α₁ and β₂ subunit expression is upregulated in HOCM in a coordinate manner.