The goal of the study was to evaluate the composition of leucocyte populations in different milk fractions as a basis on which to judge their possible role in the immune response in different compartments of the udder. The milk of one healthy quarter of nine dairy cows (SCC/quarter [les ]125000/ml; bacteriologically negative) was removed separately and during the course of milking divided into: cisternal milk (C), alveolar milk 0–25%, 25–50%, 50–75%, 75–100% (A25; A50; A75; A100, respectively) and residual milk (R). Each fraction was analysed for the main constituents, SCC and distribution of leucocyte populations and their viability. The content of fat increased steadily during milking and reached highest values in R. Protein and lactose increased from C to A25, decreased from A25 to A100 and reached their minimum in R. Na and Cl ion levels diminished from C to A25 and thereafter increased from A50 to R. Electrical Conductivity (EC) also decreased from C to A25 but remained similar within the alveolar samples and reached a minimum in R. SCC decreased from C to a minimum in A25 and increased subsequently to a significant maximum in R. Somatic cell viability increased throughout consecutive fractions with a maximum value in R. The ratio of cell populations in the various milk fractions showed a reverse trend of macrophages (M) and polymorphonuclear neutrophils (PMN). M values were highest in C while PMN levels increased to their maximum in the R fraction. The lymphocyte (L) fraction remained similar in C, A25, A50, A75 and R but was higher in A100. Proportions of L, PMN and M were, respectively, 9·3%, 38·2%, 52·3% in C, 10·9%, 64%, 25·1% in A25–A100 and 10·2%, 64·9%, 24·8% in R. Numbers of L, PMN and M in milk showed a similar pattern for all three cell types: high levels in C decreased to a minimum at A25 and increased steadily thereafter to their maxima in R. It is concluded that, for healthy quarters, M, the predominant cell type in C, are located near the teat canal, the main entrance of pathogens. Obviously they are the first immunological barriers for invading pathogens. In contrast, PMN are the most important population in the alveolar compartment. However, each leucocyte fraction had a higher concentration in C than in early alveolar fractions, thus indicating the crucial role of immune defence in the cisternal compartment.