This study reports a technique for two-photon fluorescence imaging with cellular resolution in awake, behaving mice, using a spherical treadmill to minimize motion artifacts. The apparatus combines an upright, table-mounted two-photon microscope with a spherical treadmill made of a large, air-supported Styrofoam ball. Mice with implanted cranial windows are head-restrained while their limbs rest on the ball's surface. After adaptation, mice can maneuver on the treadmill while their heads remain motionless. Image sequences show that brain motion is limited to ~2-5 μm, primarily in the focal plane. A Hidden-Markov-Model-based motion correction algorithm is used to correct residual in-plane motion, reducing the false positive error rate for cell-activity-dependent calcium transients to <5%. The technique allows for the measurement of behaviorally correlated calcium transients from large neuronal and astrocytic populations in awake mice, providing a new capability for in vivo two-photon microscopy of the mammalian brain.This study reports a technique for two-photon fluorescence imaging with cellular resolution in awake, behaving mice, using a spherical treadmill to minimize motion artifacts. The apparatus combines an upright, table-mounted two-photon microscope with a spherical treadmill made of a large, air-supported Styrofoam ball. Mice with implanted cranial windows are head-restrained while their limbs rest on the ball's surface. After adaptation, mice can maneuver on the treadmill while their heads remain motionless. Image sequences show that brain motion is limited to ~2-5 μm, primarily in the focal plane. A Hidden-Markov-Model-based motion correction algorithm is used to correct residual in-plane motion, reducing the false positive error rate for cell-activity-dependent calcium transients to <5%. The technique allows for the measurement of behaviorally correlated calcium transients from large neuronal and astrocytic populations in awake mice, providing a new capability for in vivo two-photon microscopy of the mammalian brain.