Fluorescent protein (XFP) expression in postnatal neurons allows the anatomical and physiological investigation of identified subpopulations of interneurons with established techniques. However, the spatiotemporal pattern of activity of these XFP neurons within a network and their role in the functional output of the network are more challenging issues to investigate. Here we apply two-photon excitation laser scanning microscopy to mouse spinal cord locomotor networks and present the methodology by which calcium activity can be recorded in XFP-expressing neurons. Such activity can be studied both in relation to neighboring non-XFP neurons in a spinal cord slice preparation and in relation to functional locomotor output monitored by ventral root activity in the intact in vitro spinal cord. Thus the network properties and functional correlates with locomotion of identified populations of interneurons can be studied simultaneously.