Immunotherapy and combination treatments such as biochemotherapy have shown promise, with higher response rates and a subset of durable responses; however, as the majority of responses are still of short duration, they do not provide any survival benefit. There is therefore a great need to better understand the mechanisms whereby tumours escape immune surveillance. The present study examines the expression of CD28 in patients with untreated and treated melanoma metastases. Twenty-eight patients with metastatic malignant melanoma were treated by biochemotherapy (cisplatinum 30 mg/m(2) days 1-3, DTIC 250 mg/m(2) days 1-3 i.v., and IFN-alpha2b 10 million IU s.c. three days a week for 28 days treatment cycle). Tumours were resected post-biochemotherapy and analysed for the expression of CD28 in CD4(+) and CD8(+) lymphocytes in areas where histopathological regressive changes had occurred, and close to tumour cells in areas of unaffected tumour growth using a double-staining technique. A high percentage of the lymphocytes in areas with regressive changes were found to be CD4(+)CD28(-). In contrast, the vast majority of CD4(+) lymphocytes migrating close to the tumour cells were found to be CD28(+) (P<0.001). A similar difference in the expression of CD28 was also found for the CD8(+) subset (P=0.004). A difference in down-regulation of the expression of CD28 was found between CD4(+) and CD8(+) lymphocytes both in the areas of regressive changes and in the unaffected tumour areas. The present study demonstrates that extensive down-regulation of the co-stimulatory factor CD28 is found in metastases following biochemotherapy. These results indicate that parameters of importance for the immune function have already undergone modification after one or two treatment cycles and that this down-regulation occurs in particular in areas with regressive tumour changes.