Children with spina bifida display a constellation of clinical features which include growth retardation, latex allergies, and recurrent infections. Clinical and epidemiological findings support the view that principle components of this congenital syndrome originate in an inherent susceptibility, and not necessarily as a secondary complication of the early central nervous system malformation. Critical requirements for normal neurulation include folic acid and a fully functional methylation cycle, both of which also promote cell proliferation. This suggests that elements of susceptibility may emerge as cells from an individual with myelomeningocele are induced to synthesize DNA and divide. As an in vitro correlate of proliferative activity, we compared cell cycle properties of peripheral blood lymphocytes (PBL) from children with spina bifida to those in age-matched healthy controls. The four patients selected for study all have typical lumbar level myelomeningocele with the Chiari II malformation as well as clinical features which we commonly observe. We exposed PBL to phytohemagglutinin in the presence of the thymidine analog bromodeoxyuridine. Using bivariate flow cytometric analysis of Hoechst 33258- and ethidium bromide-induced fluorescence, we measured percentages of cells which responded to the mitogen, and relative rates at which case and control cells traversed the first three cell cycles. In three of the four experiments a greater percentage of PBL from the patient than from the control responded to mitogen. Cells from these children also appeared to progress more rapidly into second and third cell cycles. In the fourth patient, an unusually high percentage of cells failed to respond to mitogen and the remainder progressed more slowly into later cycles. The biological functions of folic acid and methionine led us to expect that in the cells from children with spina bifida, DNA synthesis would be retarded, S phase prolonged and transitions between cycles delayed. These preliminary results contrast with original expectations for fractions of mitogen-responsive cells and rates of traversal. Further experiments with PBL and other cell types will be required to confirm the differences we observed and establish their biological significance. An association between abnormal proliferative capacity and NTD, whether positive or negative, will create a biological and experimental context in which to define metabolic factors in this condition.