The location of cis-acting regulatory sequences within the long terminal repeat (LTR) of the human T cell lymphotropic virus type III (HTLV-III/LAV) was determined. An enhancer element capable of increasing the rate of transcription from a heterologous promoter, irrespective of distance and orientation, is located between nucleotides -137 and -17 (cap site +1). The promoter sequences present near the TATA box respond to heterologous enhancers. The sequences present between nucleotides -17 and +80 are responsive to HTLV-III-associated trans-acting regulatory factors. Activation of these sequences by the viral regulatory factors requires the presence of a functional enhancer. The enhancer requirement is nonspecific, as the enhancer sequences of RSV, HTLV-I, and SV40 can functionally replace the HTLV-III enhancer. These findings define a new type of regulatory element, provide insight into the mechanisms that regulate HTLV-III gene expression, and may help to explain the effects of this virus on infected cells.