Staircase potentiation does not occur in atrophied rat gastrocnemius muscle subjected to repetitive stimulation. Since twitch potentiation is strongly correlated with phosphorylation of the regulatory light chains (R-LC) of myosin, our purpose was to determine if R-LC phosphorylation with repetitive stimulation is also diminished in atrophied gastrocnemius muscle. Following 2 weeks of disuse, induced by delivery of tetrodotoxin (TTX) to the sciatic nerve, phosphorylation and twitch developed tension were measured at 10 s of 10-Hz stimulation in situ in control, sham, and TTX-treated rats. Phosphorylation was significantly diminished in TTX-treated animals (9.3 +/- 3.7%), compared with control (57.0 +/- 3.4%) and sham-treated (45.5 +/- 5.6%) animals. Concomitantly, at 10 s of 10-Hz stimulation, potentiation was absent in atrophied muscles (74.4 +/- 9.4% of initial twitch), whereas control (188.4 +/- 10.3%) and sham-treated (151.7 +/- 10.3%) muscles demonstrated a great deal of potentiation. The twitch contraction of the atrophied muscles had a decreased rate of tension development and peak rate of relaxation, and an increased contraction time and half-relaxation time. The observation that the light chains are present, but not phosphorylated, suggests that the process of phosphorylation is impaired. The association of diminished phosphorylation with the absence of staircase supports the theory that R-LC phosphorylation may be the principal mechanism for twitch potentiation but does not rule out other contributing factors.