Multiple Ca2+ channel beta-subunit (Ca(v)beta) isoforms are known to differentially regulate the functional properties and membrane trafficking of high-voltage-activated Ca2+ channels, but the precise isoform expression pattern of Ca(v)beta subunits in ventricular muscle has not been fully characterized. Using sequence data from the Human Genome Project to define the intron/exon structure of the four known Ca(v)beta genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Ca(v)beta isoforms. A total of 18 different Ca(v)beta isoforms were detected in both canine and human ventricles including splice variants from all four Ca(v)beta genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Ca(v)beta subunit genes are expressed at the protein level, and the Ca(v)beta subunits show differential subcellular localization with Ca(v)beta1b, Ca(v)beta2, and Ca(v)beta3 predominantly localized to the T-tubule sarcolemma, whereas Ca(v)beta1a and Ca(v)beta4 are more prevalent in the surface sarcolemma. Coexpression of the novel Ca(v)beta2c subunits (Ca(v)beta(2cN1), Ca(v)beta(2cN2), Ca(v)beta(2cN4)) with the pore-forming alpha1C (Ca(v)1.2) and Ca(v)alpha2delta subunits in HEK 293 cells resulted in a marked increase in ionic current and Ca(v)beta2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Ca(v)beta subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca2+ channels with distinct functional properties.