During the first three months following cardiac transplantation (Tx) most recipients will experience at least one episode of acute rejection. It is known that approximately 30-40% of patients with biopsies showing Billingham grade I rejection proceed to grade III rejection that requires treatment. The category of mild rejection (Grade 1) is therefore an extremely important one. It has, however, been suggested that this grade is more important in the months immediately following Tx. Previous studies in our rat cardiac allograft model have shown that identification of markers of immune activation were indicators of poor prognosis, associated with progressive rejection. We therefore examined the type of infiltrate, and the predictive value of identifying activated cells, within serial paraffin-embedded EMBs. Serial sections <i>of 114 EMB</i> specimens ranging from grade 0 to grade 3A rejection were examined immunohistologically using monoclonal antibodies for the presence of various leucocyte populations, as well as the activation molecule, proliferating cell nuclear aitigen (PCNA, cyclin). In all grades of rejection, mononuclear cells showed labelling for CD45 (leukocyte-common antigen, >98% leucocytes), memory T cells (CD45RO, UCHL1, 43%±19% leukocytes), helper cells (OFD4, 27%±17% leukocytes) and macrophages (Mac-387, 65%±18%), and >100 EMBs showed foci of CD20+ B cells (L26). Whilst the absolute numbers of mononuclear cells (MNC) varied, the percentage of population subtypes remained constant. By contrast, the percentage of MNC labelling for PCNA was dependent on both grade of rejection and time post-Tx. During the first 3 months post-Tx, PCNA+ MNC were only seen in significant numbers during evolving rejection. PCNA labelling was maximal in biopsies which initially showed only early rejection (grade 1A, 25-30% of MNC) but then progressed to higher grades of rejection (typically 3A). EMB showing resolving (1A) rejection lacked PCNA expression, as did areas of Quilty. Higher grades of rejection (3A) were also negative for PCNA, presumably reflecting increased immunosuppressive therapy. However, by 4-5 months post-Tx, PCNA labelling in the early stages of rejection was not as marked (≤5%) as during the initial 3 month period post-Tx. These findings suggest that in the first three months after cardiac Tx, PCNA labelling of intragraft MNC is a reflection of immunologically activated inflammatory cells and, importandy, is prognostic of further rejection episodes of increasing severity if left untreated.