Ultraviolet (UV) radiation is a potent activator of human immunodeficiency virus (HIV) gene expression in a HeLa cell clone having stably integrated copies of an HIVcat (cat gene under control of the HIV promoter) reporter construct, whereas ionizing radiation is ineffective. UV-activated HIV gene expression is completely blocked by the specific p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 and by expression of a kinase-inactive p38 mutant that interferes with normal p38 function, suggesting that this stress-activated protein kinase plays an important role in UV-mediated transcriptional activation of HIV. In support of these findings, we show here that Western blot analysis demonstrated rapid and significant activation of p38 MAP kinase by UV. On the other hand, γ-radiation activated p38 MAP kinase very poorly in HeLa cells at both low and high doses at times (5–30 min) when UV radiation was effective. UV radiation also activated HIV gene expression (≤9-fold) in 1G5 Jurkat T-cells stably transfected with a luciferase reporter gene under control of the HIV promoter. In these cells, γ-radiation stimulated HIV gene expression but to a lesser extent (≤3-fold) and with different kinetics than after UV radiation, and this response was obliterated by the incubation of cells with the mitogen-activated protein kinase/Erk kinase (MEK)-½ inhibitor PD98059. This result suggests that in these cells signaling in response to γ-radiation is transduced through the MEK-½/p42/44 MAP kinase pathway to increase HIV gene expression. All combined, these results suggest that activation of p38 MAP kinase is necessary for efficient HIV gene expression triggered by DNA damaging agents, and, in a cell type-specific manner, activation of the MEK-½/p42/44 MAP kinase pathway is important for triggering a response to γ-radiation. Thus, it appears as if UV signaling leading to HIV gene expression requires the p38 MAP kinase pathway whereas activation by γ-radiation requires the MEK-½/p42/44 MAP kinase pathway.