Mycoplasmas are tiny polymorphic prokaryotic organisms (0.2-0.3 microm) that lack a cell wall and reside ubiquitously at the cell membrane or internalized into the cell. The organisms have been implicated in many diseases including functioning as cofactors catalyzing the HIV disease state. The oncogenic potential of mycoplasmas was only recently realized when they were shown to cause chromosomal changes and in vitro cell transformations through gradual progressive chromosomal loss and translocations. While a recent study linked mycoplasmas with gastric cancer, the association between mycoplasmas and ovarian cancer has not been established. Recently, a commercial assay which combined polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) methods was developed for the detection of mycoplasmas. The present objective was to determine the prevalence of mycoplasmas in archived paraffin-embedded malignant ovarian cancer tissue. The combined PCR-ELISA procedure was used with consensus primers targeting for 15 species of mycoplasmas and acholeplasmas. Archived human malignant ovarian cancer tissues (N = 27 cases) embedded in paraffin blocks were processed, and DNA was extracted and the presence of DNA verified. The extracted DNA specimens were randomly divided into three groups for analyses. PCR-ELISA assays were performed on extracted DNA together with appropriate negative and positive controls. The results showed mycoplasmas were present in 59.3% of the malignant ovarian cancer specimens. PCR-ELISA analysis of Neisseria gonorrhea and Chlamydia trachomatis controls did not produce cross-reacting false-positive results. The results suggest an association between mycoplasmas and malignant ovarian cancer. A 59.3% prevalence rate was demonstrated for mycoplasmas in paraffin-embedded ovarian cancer tissues. The mechanism involved in oncogenesis by mycoplasmas remains to be elucidated.