To examine changes in lymphocyte subpopulations in early pregnancy using a methodologically appropriate study design that addresses previous sources of bias. Thirty-seven healthy women without risk factors for human immunodeficiency virus (HIV) were reviewed when less than 9 weeks pregnant (median 51 days, range 44-61) and again at least 4 weeks following termination of pregnancy. No woman took the oral contraceptive pill. Blood was taken on each occasion at the same time of day under the same conditions of rest and food intake, transported immediately to the laboratory, and directly prepared for analysis. Lymphocyte surface markers were determined by staining with dual-colored, isotype-matched monoclonal antibody fluorescent conjugates, followed by whole blood lysis and subsequent flow cytometric analysis. Pregnancy was associated with a significant reduction in total lymphocytes (P < .0001) and also in CD4+ cells, whether expressed as a percentage of lymphocytes (P = .004), an absolute count (P = .0006), or a ratio (P = .01). Change was independent of the basal level except for lymphocytes, and almost all indices had significant correlations between pregnant and nonpregnant values. In this study design, each woman served as her own control and all factors remained constant except the pregnancy state. Early pregnancy causes a reduction in total lymphocytes of about 6% expressed as a percentage of total white cell count, and in CD4+ cells by 3% as a percentage of lymphocytes, or 100/mm3. We believe this fall can be accepted as definitive.