An anti-tumour agent 5, 6-dimethylxanthenone-4-acetic acid (5, 6-MeXAA) induced nitric oxide synthase (NOS) in the tumour, spleen, thymus and small intestine, but not in the lung, liver, kidney, heart or skeletal muscle in B6D2F1 mice bearing subcutaneous colon 38 tumours. This pattern of induction is distinct from that caused by agents such as endotoxin, muramyl dipeptide or Corynebacterium parvum. The induction of NOS (iNOS) in the tumour was more persistent (maximal at 3 days) than in other tissues (maximal at 12 h). Immunohistochemical staining suggested that iNOS was located in macrophages and endothelial cells within and around the tumour. Treatment with 5, 6-MeXAA also caused substantial increases in plasma nitrite and nitrate (NOx) concentrations that peaked at 8-12 h after 5, 6-MeXAA. The increase in plasma NOx was prevented by a NOS inhibitor N-iminoethyl-L-ornithine (L-NIO), indicating that it was due to enhanced production of NO. Tumour-bearing mice were more responsive than controls to 5, 6-MeXAA both in their plasma NOx increase and in their lower maximally tolerated dose. L-NIO was unable to prevent the complete tumour necrosis and regression caused by 5, 6-MeXAA at a dose that substantially inhibited the increase of plasma NOx. In conclusion, the experimental anti-tumour agent 5, 6-MeXAA induced NO synthesis in tumour-associated macrophages and in immunologically active tissues in parallel with its effects on tumour growth. The experiments with a non-selective NOS inhibitor L-NIO, however, suggest that NO is not a significant component in the mechanism of the anti-tumour action of 5, 6-MeXAA in this particular model.