Recent studies suggest a dual role for nitric oxide (NO) in tumour biology. High concentrations of NO can mediate tumouricidal activity, whereas lower concentrations have been shown to promote tumour growth. In this study, NO synthase (NOS) activity was investigated in cells that were prepared from tissue from primary and metastatic sites and from malignant effusions in 41 cases of suspected ovarian cancer. NO biosynthesis, determined by nitrite + nitrate (NOx) accumulation in medium from cultured cells prepared from disaggregated tumours or effusions and indicative of the inducible NO synthase isoform, was detected in 37% of the cases investigated (range 10.2-114 microM). There was a significant relationship between NOx and tumour differentiation (P = 0.014), with NOx being significantly higher for the more differentiated tumours. NOS activity, determined by the conversion of radiolabelled L-arginine to citrulline by tissue or cell extracts, was detected in 29% of cases (range 0.9-6.9 pmol/min per mg of protein), with all samples tested being moderately or poorly differentiated. Seventy percent of this activity was calcium dependent, indicative of constitutive NOS isoforms. Morphological and immunohistochemical assessment of tumour samples indicated a significant relationship between high macrophage content and NOS activity (as NOx biosynthesis) (rs = 0.726, N = 16, P < 0.01). The relationship between NOS expression, immune response, and disease progression is complex and not simply dependent on the differentiation status of ovarian cancer.