The Bcl-2 family plays an important role in tumourigenesis but its role in the genesis of canine lymphoma is yet unexplored. The aims of the present study were to select antibodies for immunohistochemical detection of various Bcl-2 family members in canine tissues and to analyse the expression of two anti-apoptotic members, Mcl-1 and Bcl-x, in normal canine and lymphoma tissues. A total of twelve antibodies out of a panel of twenty-one were deemed to specifically cross-react with canine recombinant Mcl-1, Bcl-x, Bcl-w, Bak and Bax expressed in bacteria. This was determined using an immunohistochemical method in formalin-fixed, paraffin-embedded bacteria and confirmatory Western blotting. Immunohistochemical protocols for Mcl-1 and Bcl-x were further optimized for use in canine tissues using cultured canine cells and selected normal tissues fixed in formalin and embedded in paraffin. Immunohistochemical expression patterns of these two proteins in normal canine tissues, detected using tissue arrays, are described here for the first time. They roughly coincide with the expression patterns reported in human tissues. With the same techniques, canine lymphomas were shown to frequently express Mcl-1 and, to a lesser extent, Bcl-x, across a wide range of different subtypes. These findings suggest a role for these two anti-apoptotic Bcl-2 family members in the pathogenesis of canine lymphoma.