It is generally thought that the rapid relaxation of fast muscles is facilitated by the Ca(2+) binding protein parvalbumin (Parv). Indeed superfast swimbladder (SWB) muscle of toadfish contains the largest concentration of this protein ever observed (up to 1.5 mM). At 15 degrees C toadfish perform a 100 Hz call, 400 ms in duration, followed by a long (5-15 s) intercall interval. It has been proposed that Parv helps sequester the Ca(2+) during the call, and then Ca(2+) unbinds and is pumped back into the sarcoplasmic reticulum during the long intercall interval. Midshipman (Porichthys notatus) is another fish which calls at a high frequency; 80-100 Hz at a temperature of 12-15 degrees C. However, unlike toadfish, midshipman call with a 100% duty cycle. Without an intercall interval, Parv would seem of little use as it would become saturated early in calling. Here we show that the midshipman SWB has only about 1/8th of the Parv in toadfish. Moreover, total Parv content in calling male midshipman SWB was not different from that in the non-calling female and the much slower locomotory muscles. These data suggest that Parv does not play a large role in the calling of midshipman, which is accomplished without a high concentration of this protein. Native gel-electrophoresis also revealed presence of three major (PA-I, PA-II and PA-III) and two minor (PA-Ia and PA-IIIa, <5% of total content) Parv isoforms in adult toadfish SWB. Midshipman SWB contained about equal amounts of PA-I and PA-II and also a small (approximately 10%) amount of PA-III. By amino acid composition, toadfish PA-Ia and PA-I isoforms were different from PA-II and PA-III isoforms (by 24 and 14 residues, respectively).