We previously examined the segregation patterns of two polymorphic restriction enzyme sites for EcoRI and HindIII in the 3'-flanking region of human CG-alpha gene in placenta and choriocarcinoma. We observed that a unique combination of restriction sites predominates in choriocarcinoma while this pattern was rare in placenta, suggesting a functionally important site in this region of genome. Genomic libraries were constructed from DNA derived from placenta and from the choriocarcinoma cell line JAr to examine the DNA sequence responsible for these polymorphic sites. Several clones were isolated; restriction fragments containing the polymorphic sites were subcloned into pBR322, and the stretch of DNA encompassing the polymorphic sites were sequenced. No apparent DNA rearrangements were observed; sequence analysis showed that both of the polymorphic sites were caused by single base pair mutation in the recognition sequence of each enzyme. Three new polymorphic sites were identified in the 3' region of the hCG-alpha gene. These data indicate that major DNA rearrangements in the 3' region of the gene are not associated with choriocarcinoma. However, analysis of the polymorphic sites revealed that there is a linkage disequilibrium among these sites which may extend to a putative oncogene which is responsible for developing choriocarcinoma.