Our earlier studies showed that although CD27 was stably expressed on the CD45RA(+)-CD45RO-CD29low subset of CD4 T cells, its expression on the CD45RA-CD45RO+CD29high subset of CD4 T cells was gradually lost within 3 weeks after PHA activation. In the present study, we further determined the mechanisms by which the CD27 expression was differentially regulated on the subsets of CD4 T cells. We showed that disappearance of CD27 from the surface of the CD45RA-CD45RO+ subset of CD4 T cells was not solely due to the shedding of the CD27 molecule from the cell surface since the release of a soluble form of the CD27 molecule from the CD45RO+ CD4 T cells was consistently less than that from CD45RA+ CD4 T cells. Although the surface CD27 expression was undetectable on long-term cultured T cell lines originally derived from CD45RO+ CD4 T cells, some of these cells still expressed intracellular CD27 and CD27 mRNA. Moreover, restimulation could not induce CD27 expression on such cells. Further analysis of CD27 protein and mRNA expression at a clonal level showed that cloned cells derived from CD45RO+ CD4 T cells having lost cell surface expression of CD27 were of two types: one expressed intracellular CD27 mRNA and protein whereas the second lacked both intracellular CD27 mRNA and protein.