We have previously shown that changes in the intracellular free calcium concentration ([Ca2+]i) modulate the rate of anaphase chromosome motion in stamen hair cells of Tradescantia. Elevations between 0.8-1.0-mu-M accelerate motion, while increases above 2.0-mu-M or decreases below resting level inhibit motion. The related signaling agent GTP-gamma-S also accelerates motion, however, by a mechanism that does not appear to involve changes in [Ca2+]i. To explore further the mechanism by which Ca2+ and GTP-gamma-S regulate chromosome motion we have analyzed the direct effect of these agents on the structure of the spindle microtubules (MTs). First, we injected carboxyfluorescein-derivatized brain tubulin and allowed it to incorporate into spindle MTs. Then, during appropriate times of anaphase, we injected Ca2+ or related agents and monitored their effect on spindle MT fluorescence using a confocal laser scanning microscope. A high level of Ca2+ (10-mu-M), known to inhibit motion, causes extensive degradation of spindle MT structure. An intermediate level (2-mu-M), which slows but does not stop movement, produces a distinct decay of fluorescence. A level of Ca2+ (0.8-1-mu-M) known to accelerate motion, however, generates only a small change in which the kinetochore fibers appear less distinct, and the overall spindle fluorescence is more diffuse. The Ca2+ buffer EGTA, which transiently blocks motion, has no detectable effect on spindle structure. GTP-gamma-S, which enhances motion also has no discernible effect on spindle structure. The results with Ca2+ support the idea that the ion facilitates anaphase motion through depolymerization of kinetochore MTs. However, if the depolymerization is extensive then inhibition of motion ensues. GTP-gamma-S, on the other hand, accelerates motion without inducing a detectable effect on spindle MT structure.