LY290181, 2-amino-4-(3-pyridyl)-4H-naphtho(1,2-b)pyran-3-carbonitrile, is a potent antiproliferative compound that blocks cells in the G2/M phase of the cell cycle by an apparent action on microtubules. In the present work we found that LY290181 bound to tubulin with high affinity (1 mol of LY290181 per mol of tubulin dimer; Ka, 3.8 x 10(5) M-1) and that it did not appear to bind at the colchicine or vinblastine-binding sites. LY290181 strongly stabilized microtubule dynamics as determined by video microscopy. It reduced the rate and extent of growing and shortening, reduced the catastrophe frequency and increased the rescue frequency, and increased the percentage of time the microtubules spent in an attenuated state. At the lowest effective LY290181 concentrations (0.1-0.3 microM), suppression of dynamics occurred with little reduction in polymer mass. However, at higher concentrations, LY290181 strongly reduced the polymer mass with half-maximal inhibition at a concentration of 0.75 microM. We suggest that LY290181 may exert its antiproliferative activity by stabilizing spindle microtubule dynamics by binding to tubulin at a novel site.