AbstractDespite decades of microelectrode recordings, fundamental questions remain about how auditory cortex represents sound-source location. Here, we used in vivo 2-photon calcium imaging to measure the sensitivity of layer II/III neurons in mouse primary auditory cortex (A1) to interaural level differences (ILDs), the principal spatial cue in this species. Although most ILD-sensitive neurons preferred ILDs favoring the contralateral ear, neurons with either midline or ipsilateral preferences were also present. An opponent-channel decoder accurately classified ILDs using the difference in responses between populations of neurons that preferred contralateral-ear-greater and ipsilateral-ear-greater stimuli. We also examined the spatial organization of binaural tuning properties across the imaged neurons with unprecedented resolution. Neurons driven exclusively by contralateral ear stimuli or by binaural stimulation occasionally formed local clusters, but their binaural categories and ILD preferences were not spatially organized on a more global scale. In contrast, the sound frequency preferences of most neurons within local cortical regions fell within a restricted frequency range, and a tonotopic gradient was observed across the cortical surface of individual mice. These results indicate that the representation of ILDs in mouse A1 is comparable to that of most other mammalian species, and appears to lack systematic or consistent spatial order.