The assembly of the mitotic spindle after depletion of the major gamma-tubulin isotype by RNA-mediated interference was assessed in the Drosophila S2 cell line. Depletion of gamma-tubulin had no significant effect on the cytoskeletal microtubules during interphase. However, it promoted an increase in the mitotic index, resulting mainly in monopolar and, to a lesser extent, asymmetrical bipolar prometaphases lacking astral microtubules. This mitotic accumulation coincided with the activation of the mitotic checkpoint. Immunostaining with an anti-Asp antibody revealed that the spindle poles, which were always devoid of gamma-tubulin, were unfocused and organized into sub-spindles. Despite the marked depletion of gamma-tubulin, the pericentriolar proteins CP190 and centrosomin were recruited to the spindle pole(s), where they formed three or four dots, suggesting the presence of several centrioles. Electron microscopic reconstructions demonstrated that most of the monopolar spindles exhibited three or four centrioles, indicating centriole duplication with a failure in the separation process. Most of the centrioles were shortened, suggesting a role for gamma-tubulin in centriole morphogenesis. Moreover, in contrast to metaphases observed in control cells, in which the spindle microtubules radiated from the pericentriolar material, in gamma-tubulin-depleted cells, microtubule assembly still occurred at the poles but involved the elongation of centriolar microtubule triplets. Our results demonstrate that, after depletion of gamma-tubulin, the pericentriolar material is unable to promote efficient microtubule nucleation. They point to an alternative mechanism of centrosomal microtubule assembly that contributes to the formation of abnormal, albeit partially functional, mitotic spindles.