Intracellular pH has been measured by laser microspectrofluorimetry, using the pH-sensitive dyes SNARF-1, SNARF-calcein and SNARF-1-dextran. By this technique it was possible to accurately determine pH in volumes as small as 0.5 x 0.5 x 1 microns 3. The probes were loaded into the cells either by diffusion of their acetoxymethylester derivatives (SNARF-1-AM, SNARF-calcein-AM) or by microinjection (SNARF-1-dextran). When the five types of cells were studied in RPMI medium, the nuclear pH was consistently found to be 0.3 to 0.5 units above that of the cytosol. Although the presence of pores in the nuclear membrane has been taken as evidence that free diffusion of ions and small molecules can occur in and out the nucleus, we conclude that the nuclear membrane of these cells presents a permeability barrier to H+. The pH gradient was not observed in cells suspended in PBS.