BACKGROUND: The major cause of fetal hemolytic disease is maternal immunization to D in D-incompatible pregnancies. To prevent complications, D-incompatible pregnancies are monitored for the level of maternal anti-D. At present, the monitoring of anti-D levels is performed by the indirect antiglobulin test complemented by quantitation by the technique used in an automated antibody detection and quantitation instrument. STUDY DESIGN AND METHODS: Flow cytometry was used to quantitatively determine the level of anti-D in serum and to analyze the IgG subclass distribution and the presence of IgM anti-D in these samples. The results were compared to the indirect antiglobulin test titer and to the results obtained by the technique used in an automated antibody detection and quantitation instrument. RESULTS: Flow cytometry allowed sensitive and accurate determinations of anti-D levels with low interassay and intra-assay variability, both for serum samples and standard curves. CONCLUSION: Flow cytometry is a simple, rapid, and reliable method for determining the serum levels of D antibodies and their Ig subclass distribution. It is therefore well suited for the monitoring of women during D-incompatible pregnancies.