The role of microtubules (MTs) in cell locomotion is uncertain: while MTs are not essential for motility of certain cells, MTs are necessary for the directed translocation of large cells such as fibroblasts, endothelial cells and neuronal growth cones. Based on previous studies, we hypothesize that cell locomotion may involve MTs in two possible ways: (1) the rate of cell locomotion is proportional to MT level; or (2) cell locomotion is not proportional to MT level but requires a critical level of MTs to proceed. To test these hypotheses, we measured the rate of locomotion of NRK fibroblasts migrating into an in vitro wound, before and after treatment with different concentrations of nocodazole to generate cells with different levels of MTs. Locomotion of cells was monitored directly using timelapse recording and analyzed with an Image-1 image analysis program. Addition of nocodazole (> or = 50 nM) resulted in a rapid reduction in locomotion to a new rate that was maintained for > 2 hours. We found that addition of as little as 100 nM nocodazole decreased the rate of locomotion by more than 60%; and that 300 nM nocodazole completely stopped cell locomotion. Although 100 nM nocodazole decreased locomotion over 60%, we detected no qualitative change in MT distribution by immunofluorescence. Quantitative analysis of MT fluorescence in immunofluorescently stained preparations showed that 100 nM nocodazole had no detectable effect on MT levels and that 300 nM nocodazole only decreased MT levels to approximately 40% of controls. Quantitative analysis of tubulin polymer levels by cell extraction and western blotting yielded results similar to those obtained by quantification of MT fluorescence. A comparison of the locomotion rate measurements with the MT level measurements indicated that over half of the cell locomotion rate could be blocked by nocodazole without significantly affecting MT levels in the cell; the remaining locomotion rate was reduced proportionally to MT levels. These results do not support the notion that a critical level of MTs is required for cell locomotion and suggest that only a portion (< 50%) of the speed of the cells is proportional to MT levels. Rather, by analogy with studies of MT antagonists on the mitotic spindle, they suggest a third possibility: that low concentrations of nocodazole interfere with MT dynamics and thus, MT dynamics are critical for the maximal speed of cell locomotion. This notion was further supported by analogous effects of taxol and vinblastine on cell locomotion: at concentrations that reportedly cause little change in the level of MTs, taxol and vinblastine also dramatically decreased the rate of locomotion of NRK cells. In summary, our results establish the relationship between microtubule levels and locomotion rate and suggest that dynamic MTs are rate-limiting for fibroblast locomotion.