Optimal conditions of a microenzyme-linked immunosorbent assay using a group-specific membrane antigen of Ureaplasma urealyticum serotype 7 were established with rabbit antisera and applied for the evaluation of immunoglobulin M (IgM) and IgG antibodies in 139 serum specimens from pregnant women between 26 and 38 weeks of gestation, and the assay was compared with microorganism culture and investigated to determine the role of U. urealyticum in perinatal morbidity and mortality. U. urealyticum was isolated from 75 (54%) of 139 patients; 40 had a colonization greater than or equal to 10(6) cells per ml of swab (29%); 64 (85%) of 75 culture-positive patients had IgG antibodies (absorbance mean, 0.650), versus 4 (6%) of 64 culture-negative patients (absorbance mean, 0.103) (P less than 0.001). There was no cross-reactivity with Chlamydia trachomatis infection from patients from whom no mycoplasmas were isolated, but this cross-reactivity occurred in 24% of patients with other mycoplasma infections. There was a good correlation between quantitative evaluation of U. urealyticum colonization and antibody level (P less than 0.05). However, IgM antibody was found in 30% of culture-positive patients but also in 25% of the culture-negative group. Frequency of U. urealyticum colonization was greater in unmarried young women (less than 25 years old) with a history of genital infection, and a significantly greater frequency was detected in patients who smoked (P less than 0.01) and had a lower socioeconomic status (P less than 0.001). A lower infant birth weight was more associated with U. urealyticum colonization greater than or equal to 10(5) cells per ml. The enzyme-linked immunosorbent assay provided an additional means to diagnose and evaluate U. urealyticum infection in pregnant women.