The action of the amino-reactive substances pyridoxal phosphate, 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid and 2,4,6-trinitrobenzene sulfonic acid on the contraction threshold, taken as parameter for the initiation of contraction, was investigated in fibers of the sartorius muscle of the frog. The contraction threshold was shifted by 1 to 11 mV to more negative potentials with 1 to 20 mM PDP. Similar shifts from 2 to 17 mV were produced by 0.66 to 20 mM SITS. The threshold shift was only partially reversible. The shift of the contraction threshold obtained with 2 mM SITS was nearly constant at different [Ca2+]o and [Mg2+]o from 1.5 to 50 mM with a tendency to increase at higher divalent cation concentration. TNBS had no effect on the contraction threshold. The action of PDP and SITS on the contraction threshold was successfully described by the surface charge model used earlier to explain the effect of lanthanum, neuraminidase and ruthenium red on the contraction threshold (M. Dörrscheidt-Käfer, Pfluegers Arch. 380:171-179, 181-187, 1979; J. Membrane Biol. 62:95-103, 1981). Here it was assumed that PDP and SITS bind to positive fixed charges on the surface of the T-tubular wall. This results in a shift of the calculated surface potential to more negative values which is thought to account for the measured shift of the contraction threshold.