The HIV-1 trans-activator protein Tat has been implicated as a mediator of neuronal dysfunction in several model systems. To explore the possibility that Tat can affect primary brain cells, we examined the effect of recombinant Tat protein on rat cortical brain cell cultures. Tat induced marked aggregation of neurons and astrocytes in developing cultures and caused the neuritic processes to coalesce into fascicles. Cell death was not seen and brain macrophages were not affected. These effects mapped to a different region from the trans-activation domain of Tat, as mutating the RGD (arginine-glycine-aspartic acid) sequence within the second exon abrogated aggregation and fascicle formation without affecting trans-activation capacity. Such effects on primary neurons and astrocytes may reflect specific interactions of Tat with uninfected cells within the CNS in vivo.