In visual and somatosensory cortex there are important functional differences between layers. Although it is difficult to identify laminar borders in the primary auditory cortex (AI) laminar differences in functional processing are still present. We have used electrodes inserted orthogonal to the cortical surface to compare the response properties of cells in all six layers of AI in anaesthetised guinea pigs. Cells were stimulated with short tone pips and two conspecific vocalizations. When frequency response areas were measured for 248 units the tuning bandwidth was broader for units in the deep layers. The mean Q (10) value for tuning in layers IV-VI was significantly smaller (Mann-Whitney test P < 0.001) than for layers I-III. When response latencies were measured, the shortest latencies were found in layer V and the mean latency in this layer was shorter than in any of the more superficial layers (I-IV) when compared with a Tukey analysis of variance (P < 0.005). There were also laminar differences in the best threshold with layer V having the highest mean value. The mean best threshold for layer V (32.7 dB SPL) was significantly different from the means for layers II (25.5 dB SPL) and III (26.3 dB SPL). The responses to two vocalizations also varied between layers: the response to the first phrase of a chutter was smaller and about 10 ms later in the deep layers than in layers II and III. By contrast, the response to an example of whistle was stronger in the deep layers. These results are consistent with a model of AI that involves separate inputs to different layers and descending outputs from layers V/VI (to thalamus and brainstem) that are different from the output from layers II/III (to ipsilateral cortex).