D-type cyclins are involved in regulation of cell traverse through G1 primarily by activating the cyclin-dependent kinase 4 (CDK4) and targeting it to the retinoblastoma tumour suppressor protein. There is a vast body of evidence that defective expression of D-type cyclins is associated with tumour development and/or progression. Immunocytochemical detection of D cyclins combined with multiparameter flow cytometry makes it possible to measure the expression of these proteins in individual cells in relation to their cell cycle position without the need for cell synchronization. This approach was used in the present study to compare the cell cycle phase specific expression of cyclins D3 and D1 in human normal proliferating lymphocytes and fibroblasts, respectively, with nine tumour cell lines of different lineage. During exponential, unperturbed growth, expression of cyclin D1 in fibroblasts from donors of different age, or cyclin D3 in lymphocytes, was limited to mid-G1 cells: Less than 7% of the cells entering S phase or progressing through S and G2 were cyclin D positive. In contrast, expression of either cyclin D1 or cyclin D3 in tumour cell lines of different lineage was not limited to G1 phase. Namely, over 80% of the cells in S and G2+M were cyclin D positive in eight of the nine cell lines studied. The data indicate that while expression of cyclin D1 or D3 in normal cells is discontinuous, occurring transiently in G1, these proteins are expressed in some tumour lines persistently throughout the cell cycle. This suggests that the partner kinase CDK4 is perpetually active throughout the cell cycle in these tumour lines.