The time course of phalloidin induced changes in isometric tension of partially activated skinned rabbit psoas fibres was studied as a function of both phalloidin concentration and time of pre-incubation with phalloidin. Upon addition of phalloidin to non-pretreated (control) fibres there was a fall in tension followed by an increase in tension. The latency of both parts of the response was inversely related to the phalloidin concentration in the range 40-130 microM phalloidin. By preincubating the fibres with phalloidin for varying periods of time it was possible to obtain responses which appeared to represent later portions of the control response. Thus after pre-treatment with 40 microM phalloidin in either rigor or relaxing solution for 5 min (the time corresponding to minimal tension in the control response) the tension response resembled that of the control, beginning from the vicinity of the minimum. The pattern of staining of the fibres by rhodamine-phalloidin was analysed by laser confocal microscopy to relate the mechanical response to phalloidin localization. If fibres were treated with rhodamine-phalloidin for 20-25 min there was a labelling of the I-Z-I segment with intense peaks of fluorescence at the Z-line and the ends of the I filaments. If fibres were pre-incubated for 5 min with phalloidin and then labelled with rhodamine-phalloidin the fluorescence at the Z-line and at the ends of the I filaments was suppressed and the peak of the fluorescence intensity was shifted toward the middle part of the I filament. The data indicate that the decrease in tension caused by phalloidin was associated with binding of phalloidin to the pointed ends of actin filament and the Z-line region, whereas the increase in tension occurred when phalloidin was bound along entire length of the actin filament.