To search for broadly active enhancer elements within the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, we have used a proto-enhancer amplification assay. In this assay, the enhancer region of simian virus 40 (SV40) is replaced by heterologous regulatory sequences. Upon passage in African green monkey kidney cells. SV40 growth revertants can arise by amplification (usually duplication) of active protoenhancers within the heterologous sequences. Most of the HIV-1 U3 regulatory sequences were assayed; only amplification of one or both of the HIV-1 enhancer core kappa B motifs consistently resulted in viable SV40 virus. Examination of the cell-specific enhancer activity of the individual HIV-1 kappa B proto-enhancers showed that, like the broadly active SV40 kappa B proto-enhancer (C proto-enhancer), they are all active in noninduced cell lines of either lymphoid (H9 and Jurkat) or nonlymphoid (HeLa and CV-1) origin. Unexpectedly, one of three kappa B point mutants that exhibit little or no activity in unstimulated cells is as highly induced in stimulated Jurkat cells as are the wild-type kappa B proto-enhancers. This point mutation shows that kappa B-related proto-enhancers can display markedly different activation properties in unstimulated cells yet still activate transcription to similar levels in stimulated cells.