We have developed a simple probe for certain functionally significant features of the tubulin molecule. When bovine brain tubulin is treated with N,N'-ethylene-bis(iodoacetamide) (EBI), two intrachain cross-links, designated beta s and beta *, are formed in beta-tubulin, each one with a unique effect on the electrophoretic mobility of beta on gels containing sodium dodecyl sulfate. Formation of the beta * cross-link, which involves at least one assembly-critical sulfhydryl, is completely inhibited by colchicine and its congeners, while that of beta s is inhibited completely by maytansine and GTP and partly by vinblastine. To see how conserved this complex pattern is in evolution we examined tubulins from the brine shrimp Artemia and the squid Loligo. In both tubulins EBI forms the beta * cross-link in a reaction inhibitable by colchicine, podophyllotoxin, and nocodazole. In each tubulin, EBI appears to form a second intrachain cross-link in a reaction that can be inhibited completely by maytansine and GTP and partly by vinblastine. In Artemia, this cross-link alters the electrophoretic mobility to a slightly smaller extent than is the case for beta s in bovine brain, but in Loligo the alteration is much greater. It seems that the ligand-binding sites, the critical sulfhydryls, and their spatial interrelationships are strongly conserved and that the beta s sulfhydryls or the sequence between them are less strongly conserved in evolution.