Using a combination of in vivo footprinting and site-directed mutagenesis, we have functionally characterized an enhancer located 12 kb downstream of the human immunoglobulin kappa constant-region gene. The core enhancer region is highly homologous to the murine 3' kappa enhancer. However, in addition to two regulatory elements homologous to the functional motifs of the murine enhancer, we find a third positive regulatory element in the human enhancer. This element is associated with an 11/12-bp direct repeat (DR) that is well conserved in the murine locus but was not recognized as functionally important in the murine enhancer. Mutation of any of the three motifs of the human enhancer decreases its activity to 3 to 20% of the wild-type level, indicating cooperative interaction between these elements. The DR motif does not resemble any known enhancer element and does not appear to function as a transcriptional activator on its own when present in multiple copies. Interestingly, nuclear extracts from both B- and T-cell lines contain factors binding to DR in vitro, but in vivo footprinting shows no evidence of protein-DNA binding in the T-cell line. This finding suggests that an additional regulatory mechanism, such as the effect of chromatin configuration on accessibility, may be involved in the B-cell-restricted activity of the human 3' kappa enhancer.