In the course of studying regulatory elements that affect avian embryonic rho-globin gene expression, the multipotential hematopoietic cell line K562 was transiently transfected with various rho-globin gene constructs containing or lacking an avian erythroid enhancer element. Enhanced levels of rho gene expression were seen from those constructs containing an enhancer element and minimal 5' or 3' flanking rho sequences but were not seen from enhancer-containing constructs that included extensive 5' and 3' flanking sequences. Deletion analysis localized 5' and 3' "enhancer-silencing elements" to -2140 to -2000 and +1865 to +2180 relative to the mRNA cap site. A third element required for enhancer silencing was identified within the second intron of the rho gene. The treatment of K562 cells with hemin, which induces erythroid differentiation, partially alleviated the enhancer-silencing effect. The silencer elements were able to block enhancement from a murine erythroid enhancer, but not from a nonerythroid enhancer. Electrophoretic mobility shift assays demonstrated that the transcription factor YY1 is able to bind both the 5' and 3' enhancer silencer elements; a point mutation of the single overlapping YY1/NF-Y binding site in the 3' element completely abolished the enhancer-silencing effect. These results demonstrate a complex enhancer silencer that requires 5' flanking, intronic, and 3' flanking sequences for a single regulatory effect on a eukaryotic gene.