Maintenance of blood flow is an important factor in sustaining tumour growth. Functional studies have previously demonstrated a reduction in tumour blood flow with selective inhibitors of nitric oxide (NO) synthesis, L-NAME (NG-nitro-L-arginine-methylester) and L-NMMA (NG-monomethyl-L-arginine), when administered locally to tumours derived from murine colon 26 adenocarcinoma and B16 melanoma cells. The type of NO synthase which might be responsible for this locally-derived NO and the site of synthesis was not described. Here we have investigated the distribution of immunoreactivity and the biochemical characteristics of the enzymes synthesizing NO in the same murine model. Adenocarcinoma (colon 26) or melanoma (B16) cells were introduced into a sponge matrix implanted subcutaneously in mice. After 7, 12, and 14 days, the implants were removed and frozen sections were immunostained with rabbit antisera to constitutive and inducible isoforms of NO synthase. Immunoreactivity with antisera to inducible NO synthase was detected in the vasculature of neoplastic implants, with and without the sponge, at 12 and 14 days. The enzyme was not evident in 7-day-old tumours, in non-neoplastic implants, in areas of tissue outside the tumour, or in adenocarcinoma or melanoma cells. Enzyme activity was measurable in homogenates of neoplastic implants removed at day 7 and was found to be Ca2+/calmodulin-independent. Immunoreactivity with antisera to inducible NO synthase was seen principally in the endothelium of newly-formed capillaries, identified by immunostaining for von Willebrand factor in serial sections. Immunoreactivity with antiserum to constitutive NO synthase was not evident in either neoplastic or non-neoplastic implants.(ABSTRACT TRUNCATED AT 250 WORDS)