Intron I of the rat osteocalcin gene contains silencer elements that suppress osteocalcin-reporter fusion gene transcription. The consensus sequence for the transcription factor deltaEF1 is homologous to two pyrimidine-rich repeats in intron 1 that contribute to silencing of osteocalcin-reporter fusion genes. To assess if overexpression of deltaEF1 augments transcriptional repression by these sequences, the intron 1 sequences (wtS) were placed upstream to the native rat osteocalcin promoter fused to a luciferase reporter gene (-306-OCluc). Coexpression of the wtS-(-306-OCluc) fusion gene with deltaEF1 decreased luciferase activity 30% relative to cotransfection with empty vector. Repression was abolished by point mutations in the putative deltaEF1 motifs, mS-(-306-OCluc). To determine whether deltaEF1 binds to these DNA sequences, gel retardation assays were performed using oligonucleotides containing the putative osteocalcin deltaEF1 motifs and a classical deltaEF1 motif, as radiolabeled probes. A comigrating DNA-protein complex generated by these probes was recognized by an antibody directed against deltaEF1 and competed for by excess unlabeled wild-type oligonucleotides. Oligonucleotides with mutations in the osteocalcin sequences, which abolish suppression, and in the deltaEF1 consensus site, that abolishes binding to deltaEF1, were unable to compete for the formation of this complex. Overexpression of deltaEF1 in ROS 17/2.8 cells led to an 84% decrease in osteocalcin mRNA levels relative to cells transfected with empty vector, confirming that deltaEF1 suppresses expression of the endogenous osteocalcin gene.