CD5 is a type I transmembrane protein expressed on the surface of T cells and of B1 B cells. The analysis of CD5-deficient mice suggests that CD5 can down-regulate positive signals from the antigen receptors on T and B cells but the mechanism is not known at present. In contrast to the extracellular domain the 93 amino acid long cytoplasmic domain of CD5 is highly conserved between CD5 proteins of different mammalian species. Using the yeast two-hybrid system, we identified two proteins which specifically bind to the N-terminal part of the CD5 cytoplasmic sequence. These are the Ca2+/calmodulin-dependent kinase IIdelta and Tctex-1, a light chain component of the dynein motor complex. The interaction of CD5 with the Ca2+/calmodulin-dependent kinase IIdelta was reproduced in vitro using fusion proteins. The potential function of these proteins in CD5 internalization and negative signaling is discussed.