The CD28 T cell activation pathway was functional in human tumour infiltrating lymphocytes (TIL) and can induce strong proliferation, lymphokine release and calcium mobilisation. Conversely, TIL responded poorly to stimulation via CD2, and CD28 did not synergise with CD2, which is at variance with that observed using peripheral lymphocytes from the same patients. On stimulation with anti-CD28 the monoclonal antibody, most TILs, which were CD3+, CD28+ and CD45R0+ at the beginning of culture, co-expressed both high (CD45RA) and low (CD45R0) molecular weight isoforms of CD45. CD28 was associated with the CD45R0 isoform at the cell surface of activated TIL, as demonstrated by immunoprecipitation and immunoenzymatic assay. Thus CD28 can substitute for CD3 in TIL leading to the expansion of functional lymphocytes and to the amplification of antitumour immune response.