To analyze the effects of the HIV-Tat-tubulin interaction, we microinjected HIV-Tat purified protein into Drosophila syncytial embryos. Following the Tat injection, altered timing of the cortical nuclear cycles was observed; specifically, the period between the nuclear envelope breakdown and anaphase initiation was lengthened as was the period between anaphase initiation and the formation of the next nuclear envelope. These two periods correspond to kinetochore alignment at metaphase and to mitosis exit, respectively. We also demonstrated that these two delays are the consequence of damage specifically induced by Tat on kinetochore alignment and on the timing of sister chromatid segregation at anaphase. Furthermore, we show that the expression of Tat in Drosophila larvae brain cells produces a significant percentage of polyploid and aneuploid cells. The results reported here indicate that Tat impairs the mitotic process and that Tat-tubulin interaction appears to be responsible for the observed defects. The presence of polyploid and aneuploid cells is consistent with a delay or arrest in the M phase of a substantial fraction of the cells expressing Tat, suggesting that mitotic spindle checkpoints are overridden following Tat expression.