The aim of this study was to evaluate a dual-emission fluorophore (C-SNAFL-1: 5'(and 6')-carboxyseminaphthofluorescein) for in vivo pH monitoring, using one excitation wavelength and two emissions. P388-tumour-bearing CDF mice were injected intravenously with C-SNAFL-1. Glucose was administered simultaneously to lower the tumour pH. Under our experimental conditions, tissue autofluorescence was found to be negligible. Two emission peaks were observed. The wavelength of the first one was pH insensitive (545 nm). The second one was pH sensitive: a shift of the emission peak as a function of pH was observed from 587 nm for pH 7.25 to 605 nm for pH 6.3. A calibration curve linking emission intensity ratios (I545/I635) to in vivo pH is provided. Results clearly indicate differences between tumorous (ratio 0.55 +/- 0.08, pH 6.3 +/- 0.1) and normal (ratio 1.39 +/- 0.04, pH 7.25 +/- 0.1) tissues. The concept of pH fluorescence spectroscopy in vivo using C-SNAFL-1 provides quantified data and accurate measurements of tissue pH.