Since increased muscle activity, which results in fast-slow fiber transformation, is associated with increases in sarcoplasmic Ca2+ concentration ([Ca2+]i), it seemed of interest to study the level of [Ca2+] after cessation of stimulation in fibers of the extensor digitorum longus muscle chronically stimulated (8 Hz). [Ca2+]i was measured in individual fibers with a Ca2+-sensitive electrode after subtracting the membrane potential, measured simultaneously from the potential of the Ca2+ electrode. During the first 14 days of stimulation, [Ca2+]i increased from approximately 0.1 to 0.5 microM and declined in approximately 3 wk to a value slightly higher than the initial one. The rise and decline of [Ca2+]i was preceded by a transient increase in total calcium. If stimulation was terminated after 7-8 wk when an essentially complete fast-to-slow transformation had taken place, a subsequent rest period led to a reverse slow-to-fast transformation, which was also preceded by a transient increase of [Ca2+]i reaching a peak at day 5 of rest. Unstimulated fast and slow fibers and fully transformed fibers do not differ in their [Ca2+] levels; thus it appears that the transformation process itself is accompanied, particularly in its earlier stages, by elevated [Ca2+]i levels. Elucidation of the relation between changes in Ca2+ and changes in gene expression will require further work.