Although the nonlinear optical effect known as second-harmonic generation (SHG) has been recognized since the earliest days of laser physics and was demonstrated through a microscope over 25 years ago, only in the past few years has it begun to emerge as a viable microscope imaging contrast mechanism for visualization of cell and tissue structure and function. Only small modifications are required to equip a standard laser-scanning two-photon microscope for second-harmonic imaging microscopy (SHIM). Recent studies of the three-dimensional in vivo structures of well-ordered protein assemblies, such as collagen, microtubules and muscle myosin, are beginning to establish SHIM as a nondestructive imaging modality that holds promise for both basic research and clinical pathology. Thus far the best signals have been obtained in a transmitted light geometry that precludes in vivo measurements on large living animals. This drawback may be addressed through improvements in the collection of SHG signals via an epi-illumination microscope configuration. In addition, SHG signals from certain membrane-bound dyes have been shown to be highly sensitive to membrane potential. Although this indicates that SHIM may become a valuable tool for probing cell physiology, the small signal size would limit the number of photons that could be collected during the course of a fast action potential. Better dyes and optimized microscope optics could ultimately lead to the imaging of neuronal electrical activity with SHIM.