CD97 is a newly identified, activation‐associated human leucocyte antigen with seven putative transmembrane domains. It has an extended extracellular segment containing several adhesion molecule structure motifs, and has been shown to interact with the human complement regulator, decay‐accelerating factor (DAF, CD55). To understand further the interaction between CD97 and DAF, as well as the structure and function of CD97 in general, we have cloned the mouse CD97 cDNA and studied the encoded protein for its membrane association property and ability to interact specifically with the murine decay‐accelerating factor. The full‐length mouse CD97 cDNA that we have cloned and characterized encodes a protein that is 60% identical to the three epidermal growth factor (EGF) domain‐containing form of human CD97 but does not contain the Arg‐Gly‐Asp (RGD) motif which is present in human CD97. Two other alternatively spliced forms of mouse CD97 were also identified. These forms differ by the number of EGF‐like sequence repeats present in the N‐terminal region. Northern blot analysis revealed that CD97 is expressed widely in mouse tissues and in resting as well as activated cultured mouse splenocytes. Transient transfection of human embryonic kidney (HEK) 293 cells with the mouse CD97 cDNA in a green‐fluorescence protein vector (pEGFP‐N1) showed plasma membrane targeting of the expressed protein. Western blot analysis confirmed its membrane association and identified the existence of a processed C‐terminal fragment, supporting the notion that CD97 on the cell membrane is composed of post‐translationally generated subunits. Adhesion studies demonstrated that normal, but not DAF knockout mouse erythrocytes and splenocytes adhered to mouse CD97‐transfected HEK cells. The interaction of CD97 and DAF was found to be species‐restrictive in that human erythrocytes were unable to bind to mouse CD97‐transfected HEK cells. These results indicate that the general structure, membrane association property and DAF‐binding ability of CD97 are conserved and that the adhesive interaction between CD97 and DAF is independent of the RGD motif. The finding that CD97 is distributed widely among various mouse tissues suggests that CD97 may have other roles beyond lymphocyte activation.