The new intracellular pH (pH(i)) dye carboxy-seminaphthorhodafluor (SNARF-1) was compared to the established dye 2,3-dicyanohydroquinone (DCH) using flow cytometry. Both dyes give high-resolution pH(i) measurements. SNARF-1 remains trapped within cells much longer than DCH, so that pH(i) can be monitored during and after treatments with chemicals or hyperthermia. The toxicity of the dyes is similar, and both dyes can be used at concentrations that result in low toxicity to cells. Adequate staining of cells with SNARF-1 is dependent on the cell concentration. The absolute pHi values indicated by SNARF-1 are higher than values measured with DCH. However, the trends measured by both dyes are consistent, and both are useful for making pH(i) measurements.