We have examined the role of stress-activated p38 MAP kinase in regulating human immunodeficiency virus (HIV) gene expression in response to ultraviolet light (UV). We found that UV activated p38 in HeLa cells harboring stably integrated copies of an HIVcat plasmid to levels similar to those obtained by hyperosmotic shock. However, hyperosmotic shock resulted in one order of magnitude smaller increase in CAT activity than treatment with UV. The specific p38 inhibitor SB203580 significantly decreased (>80%) UV activation of HIV gene expression whereas PD98059, a specific MEK-1 inhibitor did not, suggesting that p38 is specifically involved in the HIV UV response and little to no contribution is provided by MEK-1 and the p42/p44 MAP kinase pathway. Whereas increased binding of NF-kappaB to an oligonucleotide spanning the HIV enhancer was observed after UV, as expected, this binding was not affected by SB203580. Furthermore, UV activation of HIV gene expression in cells having the cat reporter gene under control of an HIV promoter deleted of the enhancer (-69/+80) produced results indistinguishable from those using HIVcat/HeLa cells with an intact HIV promoter (-485/+80), suggesting that SB203580 acts through the basal transcription machinery. Northern blot analysis of steady-state RNA from HIVcat/HeLa cells revealed an almost complete inhibition of UV activation with SB203580 at the RNA level. Similarly, the UV response was almost completely obliterated at the CAT and RNA levels in HIVcat/HeLa cells stably transfected with a plasmid expressing a kinase-inactive mutant of p38 (isoform alpha), without affecting NF-kappaB activation, providing strong genetic evidence that p38, at least the alpha isoform, is necessary for UV activation of HIV gene expression and that NF-kappaB activation alone is insufficient. These results firmly establish p38 MAP kinase as a key modulator of HIV gene expression in response to UV that acts independently of NF-kappaB.