The fluorescence ratio imaging technique is currently applied in vitro and in vivo to measure intracellular as well as extracellular pH. To date, however, no effort has been made to assess the technique's ability to detect spatial pH gradients in vitro or in vivo, where pH gradients are known to exist, in a controlled environment using an independent pH measurement system. We present here both in vitro calibration and in vivo application of fluorescence ratio imaging to measure pH gradients in a biologically useful pH range (6.2-7.8). Isoelectric focusing in short polyacrylamide gels was used to establish spatial pH gradients ranging from approximately 0.2 to 0.5 pH units/mm. The pH gradients were measured independently with fluorescence ratio imaging microscopy (FRIM) and a surface electrode. The average difference between measurements using the two techniques was 0.043 pH units for 10-mm polyacrylamide gels (100 measurements in 12 gels) and 0.046 pH units for 20-mm gels (114 measurements in 15 gels). These gel studies have demonstrated the use of isoelectric focusing to calibrate FRIM for measuring spatial pH gradients characteristic of those found in tumors in vivo. The feasibility of FRIM was tested in vivo by measuring pH in a solid tumor (VX2 carcinoma) and surrounding normal granulation tissue grown in the rabbit ear chamber. The average (+/- SD) pH of normal and neoplastic regions of the composite tissue was 7.18 +/- 0.11 (n = 158) and 6.75 +/- 0.10 (n = 191), respectively. The pH varied by an average of approximately 0.6 pH units over the 5-mm diameter of the chamber and showed a sharp difference at the interface of the two tissue types. These pH values are consistent with the current pH data on normal and neoplastic tissues. Thus FRIM should permit noninvasive in vivo evaluation of new pH-modifying agents and, in combination with other optical techniques, should offer novel mechanistic information about tumor pathophysiology.