Neural tissue is organized in three-dimensional (3D) networks of neuronal and glial cell populations. To understand the functional organization of these networks, it is desirable to achieve 3D activity measurements from large cell populations in intact tissue with high temporal resolution. Repeated acquisition of image stacks with standard laser-scanning microscopes is too slow. This protocol describes fast 3D calcium imaging in the living brain using mechanical laser scanning with standard galvanometric mirrors and a piezoelectric focusing element. The purpose of 3D laser scanning is to create a 3D line scan that samples relatively homogenously from a particular observation volume. The spatial resolution of this approach is low, except along the line. However, the main goal is not to resolve subcellular structures, but rather to hit as many cell bodies as possible within the volume. In this manner, local network activity can be inferred from the somatic calcium signals of a significant fraction of the cell population. With a sinusoidal swinging microscope objective as a constraint, 3D scan trajectories are generated that sample fluorescence signals from the majority of cells within a cuboidal volume. Measurements with 10-Hz temporal resolution can be achieved for population calcium signals from several hundreds of identified neurons and glial cells within cuboids with side lengths of ∼250 µm. An example cellular 3D orientation map of the rat visual cortex is presented. This 3D laser-scanning technique enables direct observation of in vivo neural network dynamics in cell populations of substantial size.