BACKGROUND The process of maturation of the immune system leads to generation of various lymphoid cell populations having the ability to react in specific way and expressing various markers on the cell surface. The study was set up to establish reference values for B lymphocyte subpopulations in peripheral blood of children and young adults to find the spectrum of their physiological age-related variation. METHODS Blood samples were taken from 292 children and young adults aged 0-31 years and tested for distribution of B cell subsets. Relative and absolute sizes of non-memory and memory, transitional, naïve, immature marginal zone-like/IgM-only memory, class-switched memory, double negative, activated, and plasmacytoid cell populations were determined by four-color flow cytometry, based on differential expression of CD19, IgM, IgD, CD21, CD27, and CD38. Significant variation both in relative, as well as in absolute numbers of individual cell populations in tested groups was observed. RESULTS The reference values for age-related B cell subsets in eleven age groups, established as result of this study, may be used in diagnostics of any pathology related to B cell maturation process, as well as in attempts of correlating laboratory results with clinical symptoms of many defects affecting antibody production in pediatric population. CONCLUSIONS Determination of B cell subpopulations carried in patients with antibody deficiencies may help to understand the nature of the disease and prevent its complications.