Multiple Ca²⁺ channel β-subunit (Ca_vβ) 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_vβ 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_vβ genes, we designed a systematic RT-PCR strategy to screen human and canine left ventricular myocardial samples for all known Ca_vβ isoforms. A total of 18 different Ca_vβ isoforms were detected in both canine and human ventricles including splice variants from all four Ca_vβ genes. Six of these isoforms have not previously been described. Western blots of ventricular membrane fractions and immunocytochemistry demonstrated that all four Ca_vβ subunit genes are expressed at the protein level, and the Ca_vβ subunits show differential subcellular localization with Ca_vβ_1b, Ca_vβ₂, and Ca_vβ₃ predominantly localized to the T-tubule sarcolemma, whereas Ca_vβ_1a and Ca_vβ₄ are more prevalent in the surface sarcolemma. Coexpression of the novel Ca_vβ_2c subunits (Ca_vβ_2cN1, Ca_vβ_2cN2, Ca_vβ_2cN4) with the pore-forming α_1C (Ca_v1.2) and Ca_vα₂δ subunits in HEK 293 cells resulted in a marked increase in ionic current and Ca_vβ_2c isoform-specific modulation of voltage-dependent activation. These results demonstrate a previously unappreciated heterogeneity of Ca_vβ subunit isoforms in ventricular myocytes and suggest the presence of different subcellular populations of Ca²⁺ channels with distinct functional properties.