Tumour pH plays a significant role in cancer treatment. However, because of the limitations of the current measurement techniques, spatially and temporally resolved pH data, obtained non-invasively in solid tumours, are not available. Fluorescence ratio imaging microscopy (FRIM) has been used previously for noninvasive, dynamic evaluation of pH in neoplastic tissue in vivo (Martin GR, Jain RK 1994, Cancer Res., 54, 5670-5674). However, owing to problems associated with quantitative fluorescence in thick biological tissues, these studies were limited to thin (50 microns) tumours. We, therefore, adapted the FRIM technique for pH determination in thick (approximately 2 mm) solid tumours in vivo using a pinhole illumination-optical sectioning (PIOS) method. Results show that (1) steep interstitial pH gradients (5 microns resolution), with different spatial patterns, exist between tumour blood vessels; (2) pH decreased by an average of 0.10 pH units over a distance of 40 microns away from the blood vessel wall, and by 0.33 pH units over a 70 microns distance; (3) the maximum pH drop, defined as the pH difference between the intervessel midpoint and the vessel wall, was positively correlated with the intervessel distance; (4) 45 min following a systemic glucose injection (6 g kg-1 i.v), interstitial pH gradients were shifted to lower pH values by an average of 0.15 pH units, while the spatial gradient (slope) was maintained, when compared with preglucose values. This pH decrease was not accompanied by significant changes in local blood flow. pH gradients returned to near-baseline values 90 min after glucose injection; (5) interstitial tumour pH before hyperglycaemia and the glucose-induced pH drop strongly depended on the local vessel density; and (6) sodium bicarbonate treatment, either acute (1 M, 0.119 ml h-1 for 3 h i.v.) or chronic (1% in drinking water for 8 days), did not significantly change interstitial tumour pH. Modified FRIM may be combined with other optical methods (e.g. phosphorescence quenching) to evaluate non-invasively the spatial and temporal characteristics of extracellular pH, intracellular pH and pO2 in solid tumours. This will offer unique information about tumour metabolism and its modification by treatment modalities used in different cancer therapies.