Voltage-activated Ca(2+) channels comprise complexes of a pore-forming Ca(V)alpha(1) and auxiliary subunits Ca(V)beta, Ca(V)alpha(2)delta and sometimes Ca(V)gamma. The intracellular Ca(V)beta subunit assists in trafficking and surface expression of the Ca(V)alpha(1) subunit and can modulate biophysical properties of the Ca(2+) channel. Four genes, Ca(V)beta1-4, exist which confer different properties to Ca(2+) currents through the various Ca(V)alpha(1) subunits. Ca(2+) currents in cochlear inner (IHC) and outer hair cells (OHC) serving synaptic transmission flow predominantly through the L-type Ca(V)alpha(1) subunit Ca(V)1.3, but associated Ca(V)beta subunits are unknown. In the organ of Corti, we found mRNA and protein for all four Ca(V)beta subunits including Ca(V)beta2, but clear assignment of the Ca(V)beta1-4 immunolabelling with hair cells or nerve fibers was difficult. We analyzed Ca(V)beta3 knockout (Ca(V)beta3(-/-)) and Ca(V)beta4 mutant mice (Ca(V)beta4(lh/lh)), which had normal hearing. Recording voltage-activated Ba(2+) currents from hair cells of the two mouse models revealed distinct significant changes of cell size and Ba(2+) current properties compared with their wild-type controls. Neonatal Ca(V)beta4(lh/lh) IHCs showed reduced membrane capacitances and changes in the voltage dependence and kinetics of current activation, whereas mature IHCs had reduced peak currents compared with Ca(V)beta4(wt), altogether indicating the presence of Ca(V)beta4 in IHCs. Ba(2+) currents of Ca(V)beta3(-/-) OHCs showed largely reduced amplitudes, changes in the voltage dependence and kinetics of Ba(2+) current activation, and increased inactivation compared with Ca(V)beta3(wt), pointing to a role of Ca(V)beta3 for OHCs. These results indicate that neither Ca(V)beta3 nor Ca(V)beta4 are indispensable for hair cell Ca(2+) currents but contribute to the overall current properties.