The cause of most microcephaly is unknown. Hanshaw's observation that 44% of microcephalics had cytomegalovirus (CMV) complement fixing (CF) antibody (Ab) suggested that inapparent congenital CMV (and other) infections might be a significant cause. Accordingly, sera from non-institutionalized microcephalic, mentally retarded normocephalic, and normal children were tested for cytomegalovirus and herpes simplex virus (HSV) complement fixing, rubella hemagglutination inhibition (HI), and toxoplasma dye test antibodies in an effort to determine whether there was a significant association between infection with these agents and microcephaly or mental retardation. Eight of 62 (13%) microcephalic children who were from 5 months to 5 years of age had titers of CMV CF Ab equal to or greater than 1:10. Four of 26 (15%) mentally retarded normocephalic children were seropositive to CMV, and two of 44 (5%) normal normocephalic children were seropositive. These differences are not statistically significant. In the age range 5 months to 5 years, 9 of 62 (15%) microcephalic, 9 of 26 (15%) normocephalic mentally retarded, and 4 of 44 (9%) normal normocephalic children were seropositive to HSV CF Ab. In the group 5 months to 5 years old tested for rubella HI Ab, 11 or 61 (18%) microcephalic, 2 of 25 (8%) normocephalic mentally retarded, and 6 of 38 (16%) normal normocephalic children were found to be seropositive. Three of 61 (5%) microcephalic, 2 of 26 (8%) mentally retarded normocephalic, and 3 of 42 (7%) normal normocephalic children from 5 months to 5 years had Ab to toxoplasma. There was no statistically significant association of evidence of infection with these three organisms and microcephaly. The authors speculate that inapparent congenital CMV infection can cause microcephaly and mental retardation without microcephaly, but that this infection causes only a small proportion of these conditions.