Processing in cortical circuits is driven by combinations of cortical and subcortical inputs. These inputs are often conceptually categorized as bottom-up, conveying sensory information, and top-down, conveying contextual information. Using intracellular recordings in mouse primary visual cortex, we measured neuronal responses to visual input, locomotion, and visuomotor mismatches. We show that layer 2/3 (L2/3) neurons compute a difference between top-down motor-related input and bottom-up visual flow input. Most L2/3 neurons responded to visuomotor mismatch with either hyperpolarization or depolarization, and the size of this response was correlated with distinct physiological properties. Consistent with a subtraction of bottom-up and top-down input, visual and motor-related inputs had opposing influence on L2/3 neurons. In infragranular neurons, we found no evidence of a difference computation and responses were consistent with positive integration of visuomotor inputs. Our results provide evidence that L2/3 functions as a bidirectional comparator of top-down and bottom-up input. •Layer 2/3 neurons show widespread subthreshold mismatch responses•Mismatch response sign is predicted by visual flow and locomotion-related responses•Layer 5/6 has a scarcity of depolarizing mismatch responses•Visual flow and locomotion speed have opposing signs of influence only in layer 2/3 Jordan and Keller use whole cell recordings in mice navigating in virtual reality to show that neurons only in superficial cortical layers have a special property: they integrate visual flow and locomotion speed with opposing signs, allowing them to compute bidirectional mismatches between actual and expected visual flow speeds.