Isolated longitudinal muscle from guinea-pig ileum was immersed in a calcium-depleted, high-potassium medium for 10 min and then induced to contract by adding 1.8 mM CaCl2. It exhibited a biphasic response that consisted of an initial, rapid, transitory contraction--the phasic response--followed by a slower more sustained contraction--the tonic response. 45Ca uptake was also measured. Only that fraction of the accumulated calcium that could be inhibited by cobalt ion was considered to be intracellular calcium. The data showed that the phasic contraction was accompanied by a brief, rapid uptake of calcium ions and the tonic response was accompanied by a slower, more prolonged uptake of calcium ions. When the muscle, after undergoing an initial biphasic contraction, was reincubated in the calcium-depleted, high-potassium medium for 70 to 80 min, the phasic contraction that it exhibited upon the readdition of 1.8 mM CaCl2 remained unaltered, but the tonic contraction was delayed and substantially diminished. Under these conditions, the rate of the initial brief, rapid uptake of calcium was reduced by 20%, whereas the subsequent slow, prolonged uptake of calcium was measurably delayed and its rate was reduced by an average of 31.4%. Calcium pulse experiments showed that variations in the rate of calcium uptake were due primarily to variations in the rate of the inward movement of calcium ions. The results suggest that the biphasic mechanical response is a reflection of a biphasic influx of calcium ions in the potassium-depolarized longitudinal muscle.