We have previously reported the identification of a naturally occurring junction between alpha satellite and satellite III DNA on human chromosomes 13, 14 and 21. Direct sequence analysis has shown that the 9 bp alphoid-derived direct repeat sequence (GTGAAAAAG) present at the junction is fully conserved on these chromosomes. A novel protein, pJ alpha, present in HeLa nuclear extracts, binds to the conserved junction sequence. Mutation analysis of the binding site suggests that pJ alpha can recognize one of the two 9 bp repeats and provides some insight into nucleotides that are important for binding. Competition studies support the possibility that this protein binds a significant portion of genomic alpha satellite DNA. Preliminary protein purification experiments have shown that pJ alpha has a molecular weight of 10-15 kDa.