We have synthesized a new photoreactive vinblastine derivative, 3-[[2-amino(4-azido-2-nitrophenyl)ethyl]-amino)-carbonyl)-O4-deceatyl -3-de (methoxycarbonyl)-vincaleukoblastine (NAPAVIN), which can be photoactivated with light in the 455-nm region as well as with ultraviolet irradiation. Previous studies had shown that photoactivated NAPAVIN is much more effective than vinblastine in inhibiting cell proliferation of multidrug resistant cell lines. The experiments reported here demonstrate that the unirradiated derivative is very similar to vinblastine in its interactions with brain tubulin and microtubules, regarding inhibition of in vitro assembly, binding, aggregation, and production of protofilament spirals. Irradiation of [3H]NAPAVIN in the presence of tubulin led to covalent binding of the drug to both subunits of the protein. Labeling also occurred when NAPAVIN was first irradiated, then incubated with tubulin in the dark, indicating the production of a fairly stable reactive species with a half-life of about 400 min. We conclude that labeling by this compound, under some conditions, occurs not by a nitrene but by an electrophilic photoproduct.