Recent advances in optical-sectioning microscopy, along with novel fluorescent proteins and probes, give us the tools to image molecules and their interactions in space and time. Investigators using these tools routinely collect multichannel three-dimensional (3D) images and time series, but analyzing such complex datasets requires sophisticated visualization techniques. We here provide an overview of the principles and practices of 3D visualization of multichannel microscopic data. We also describe ImageSurfer, a new software package for volume visualization and data analysis. ImageSurfer is freely available (www.imagesurfer.org) and provides powerful interactive tools to explore and analyze complex multichannel 3D datasets. Although ImageSurfer is designed with fluorescent microscopy in mind, it is also effective for other types of data, including 3D datasets acquired by functional magnetic resonance imaging and EM tomography.