To further assess the mechanism for the induction of anti-DNA antibodies, the response of BALB/c mice to immunization with single-stranded DNA of various species origin was determined. Anti-DNA levels of mice immunized with Escherichia coli DNA as complexes with methylated BSA in adjuvant were significantly greater by ELISA than those from mice immunized similarly with calf thymus DNA. Furthermore, comparison of the responses of mice immunized with complexes of DNA from calf thymus, chicken blood, Clostridium perfringens, E. coli, human placenta, or salmon testes indicated that the bacterial DNAs induced the highest antibody levels. The antibody response to E. coli DNA was shown by inhibition ELISA to have two populations, one binding unique determinants in E. coli DNA and the other cross-reactive with determinants expressed on all DNAs tested. These results indicate that DNA molecules, when complexed to a protein carrier, differ in their immunogenic potential, likely because of the presence of unique sequences or structures rarely presented by mammalian host DNA.