We describe a laser-scanning two-photon fluorescence microscope that is capable of observing single molecules with excellent temporal resolution and three-dimensional spatial resolution. To demonstrate the capabilities of the instrument we present. single-molecule fluorescence data obtained in several different scanning modes. In addition, a polarization-sensitive detection scheme can provide detailed three-dimensional information about the orientations of molecules in any of these scanning modes. (C) 1999 Optical Society of America OCIS codes: 180.2520, 190.4180.