We studied the steady-state rate staircase in isolated rat ventricular cells stimulated at frequencies ranging up to 6 Hz at 37 degrees C. When contractility was measured as displacement of the cell edge, amplitude of motion decreased while stimulation rates were increased from rest to 1 Hz (negative staircase). With further increases in the rate of stimulation to 6 Hz, amplitude of motion increased for each increment in stimulation rate (positive staircase). Ryanodine at a concentration of 100 nM was a negative inotrope and abolished the rested state contraction and the negative staircase seen between rest and 1 Hz (P less than 0.05) but had no significant effect on twitch amplitude or the staircase at stimulation rates between 1 and 6 Hz. L-type Ca2+ channel inhibition with verapamil (0.2 microM) had little effect on twitch or the negative staircase from rest to 1 Hz, but twitch amplitude was diminished and the staircase was converted from positive to negative at stimulation rates between 1 and 6 Hz (P less than 0.05). We conclude that in rat ventricular cells studied at 37 degrees C, the rate staircase is biphasic: negative from rest to 1 Hz and positive from 1 to 6 Hz. This phenomenon is dominated by Ca2+ release from the sarcoplasmic reticulum at rates less than 1 Hz and by Ca2+ entry through L channels at rates greater than 1 Hz.