The Ca2+-activated bioluminescent protein aequorin was used to observe intracellular [Ca2+] transients in voltage clamped canine Purkinje fibers. The pattern of luminescence during a voltage clamp pulse was characterized by two components: L1, which is a rapid initial increase in luminescence and L2, which is a slower, secondary rise of variable configuration. 1. L1, L2, inward current, and contraction were abolished by D 600 (2 microM). 2. Paired clamp pulses. L1 reprimes more rapidly than L2; L1 reprimes within 100 ms, L2 does not. 3. Clamp pulse duration. Peak inward current was the same for 50 ms or 500 ms clamp pulses; L1 was either the same or slightly reduced in 50 ms clamp pulses compared to 500 ms clamp pulses. L2, however, was abolished in repetitively given 50 ms pulses compared compared to repetitively given 500 ms pulses. When 500 ms pulses were alternated with 50 ms pulses, L2 was greater in the 50 ms pulse than in the 500 ms pulse. 4. Clamp pulse potential. In the range-35 to O mV, peak L1 and peak inward current occurred at nearly the same time, had the same threshold potential, and had a similar dependence on membrane potential. In the presence of L2, contractions develop severalfold greater peak tension, time to peak tension is longer, and relaxation is more rapid than in the absence of L2. It is concluded that Ca2+ released from stores accounts for L2 and most of the 'activator calcium'. Ca2+ from another source accounts for L1 and activates a small early component of the contraction. L1 has some properties expected for a signal related to Ca2+ entering via slow inward current, but not via Na/Ca exchange.