The human foamy or spumaretrovirus (HFV) is a complex retrovirus that codes for the three retroviral genes gag, pol, and env and the regulatory and accessory bel genes. A particular feature of HFV gene expression was recently described: not only does the HFV provirus contain the classical retroviral long terminal repeat promoter, a second functionally active promoter is present in the env gene upstream of the bel genes (M. Löchelt, W. Muranyi, and R. M. Flügel, 1993, Proc. Natl. Acad. Sci. USA 90, 7317-7321). Both, the HFV long terminal repeat promoter I and internal promoter II depend upon the HFV transcriptional transactivator Bel 1 for efficient gene expression. The internal promoter directs the synthesis of functionally active Bel 1 transactivator and Bet proteins that are expressed early after HFV infection. In this report, it is shown that mutation of the promoter II TATA box resulted in HFV proviral clones with a reduction in infectivity by a factor of approximately 100. Gene expression by promoter II TATA box mutant HFV proviruses was reduced. HFV proviruses with the mutated promoter II TATA box used cryptic start sites of transcription upstream of the original promoter II TATA box, resulting in an inefficient and less accurate transcriptional initiation. The reduced HFV structural gene expression by the mutated HFV proviruses was relieved by providing Bel 1 protein in trans. This demonstrates that HFV promoter II-directed Bel 1 expression is important for producing the high levels of Bel 1 that increases virus replication.