Human neutrophilic polymorphonuclear leukocytes (neutrophils) adhering to a substratum undergo a dramatic change in cellular morphology when treated with the tumor promoter 12-0-tetradecanoylphorbol-13-acetate (TPA). Within a few minutes of TPA treatment, the cells cease locomotion and spread symmetrically on the substratum. Concomitantly, TPA initiates centrosome splitting in a manner similar to that induced by treatment of randomly locomoting cells with a chemotactic factor. The two centrioles of a centrosome separate by a distance of several micrometers, and each of the solitary centrioles is surrounded by an aster of microtubules. Some cells also establish a third, centriole-free aster of microtubules. TPA treatment increases the total number of microtubules associated with the centrosome(s) and also increases overall polymer length. The frequency of centrosome splitting is enhanced transiently by treatment with the synthetic chemotactic peptide f-Met-Leu-Phe. Centrosome splitting is interpreted in terms of an interaction between the cell periphery and the microtubule system. Possible cellular mechanisms of this unusual phenomenon are considered.