The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.