[Arginine]vasopressin (AVP) is a neuropeptide physiologically synthesized in the hypothalamus but pathologically expressed by small-cell lung cancer (SCLC). A minimal 65 bp AVP promoter can restrict basal activity to SCLC in vitro, but a 199 bp fragment directs 5-fold higher expression in SCLC [Coulson, Stanley and Woll (1999) Br. J. Cancer 80, 1935-1944]. Several predicted E-box motifs occur within the 199 bp fragment, and we now describe an enhancer which contributes to AVP promoter tumour-specificity in some cell lines. The deletion of two adjacent E-boxes (-157 to -131) resulted in an approx. 70% loss of reporter gene expression in a SCLC line (Lu-165) with high endogenous AVP production. Using a series of AVP promoter deletion constructs and site-directed mutagenesis, we show that both these E-box sites were required for enhancer function, whereas mutation of an adjacent AP-1 site had no effect on the promoter activity. Electrophoretic-mobility-shift analysis indicated that, although both the predicted E-box motifs bound specific complexes, only one appeared to function as a strong E-box which binds basic helix-loop-helix (bHLH) factors. This motif formed a complex in lung tumour-cell extracts, which was particularly strongly bound in Lu-165, and was competed for by a characterized E-box motif from the preprotachykinin A promoter. Antibody supershifts indicate that this complex is a heterodimer of upstream stimulatory factor (USF)-1 and USF-2. Non-bHLH complexes weakly bound the second potential E-box motif in a SCLC-specific manner. These complexes were not recognized by the bHLH antibodies and remain unidentified; however, they were detected in seven of eight SCLC cell lines and not in four control lines. We postulate that there is a co-operative and complex interaction between an E-box and an adjacent site constituting a SCLC-specific enhancer within the AVP proximal promoter.