The rational development of new antineoplastic agents directed against tubulin, a protein critical for cell division, requires greater understanding of the interaction between the polypeptide subunits of tubulin, its two tightly bound guanine nucleotides, and microtubule-associated proteins (MAPs). The effects of nucleotides on the stability of microtubules continued to be examined, as were conditions to optimize the separation of alpha-tubulin and beta-tubulin on a preparative scale. The purification of a microtubule-associated protein which causes the formation of microtubule bundles continued to progress, and a project to introduce potentially antimitotic nucleotide analogs into cells continued. Roles of divalent cations in nucleotide binding to tubulin, in tubulin polymerization and in polymer stability were examined. In particular, major differences in effects of Mg2+ and Be2+ on tubulin polymerization, tubulin precipitation, polymer stability, and nucleotide binding and hydrolysis were evaluated in detail. Additional cations were also evaluated for effects on these reactions. A project to define the disulfide bridges in tubulin and in two major classes of MAPs was completed, with the observation that all cysteine sulfhydryl groups in these proteins were free. Studies were initiated to determine whether free sulfhydryls played a role in the stability of either tubulin or MAPs. We have succeeded in synthesizing 2',3'-dideoxyguanosine 5'-[alpha, beta-methylene]-triphosphate, and we are evaluating its effects on microtubule assembly.