Microtubules (MTs) contribute to the directional locomotion of many cell types through an unknown mechanism. Previously, we showed that low concentrations (<200 nM) of nocodazole or taxol reduced the rate of locomotion of NRK fibroblasts over 60% without altering MT polymer level [Liao et al., 1995: J. Cell Sci. 108:3473-3483]. In this paper, we directly measured the dynamics of MTs in migrating NRK cells injected with rhodamine tubulin and treated with low concentrations of nocodazole or taxol. Both drug treatments caused statistically significant reductions (approx. twofold) in growth and shortening rates and less dramatic effects on rescue and catastrophe transition frequencies. The percent time MTs were inactive (i.e., paused) increased greater than twofold in nocodazole- and taxol-treated cells, while the percent time growing was substantially reduced. Three parameters of MT dynamics were linearly related to the rates of locomotion determined previously: rate of shortening, percent time pausing and percent time growing. The number of MTs that came within 1 microm of the leading edge was reduced in drug-treated cells, suggesting that reduced MT dynamics may affect actin arrays necessary for cell locomotion. We examined two such structures, lamellipodium and adhesion plaques, and found that lamellipodia area was coordinately reduced with MT dynamics. No effect was detected on adhesion plaque density or distribution. In time-lapse recordings, MTs did not penetrate into the lamellipodium of untreated cells, suggesting that MTs affect lamellipodia either through their interaction with factors at the base of the lamellipodium or by releasing factors that diffuse into the lamellipodia. In support of the latter hypothesis, when all MTs were rapidly depolymerized by 20 microM nocodazole, we detected the rapid formation of exaggerated protrusions from the leading edge of the cell. Our results show for the first time a linear relationship between MT dynamics and the formation of the lamellipodium and support the idea that MT dynamics may contribute to cell locomotion by regulating the size of the lamellipodium, perhaps through diffusable factors.