Objective: Sleep is necessary for normal brain function. The neuromodulator adenosine is a key regulator of sleep homeostasis but surprisingly little is known about effects of adenosine at the level of single neurons or microcircuits. Therefore, our aim is to investigate the effects of adenosine at the level of single neurons and synaptic microcircuits. Methods: Intrinsic cell properties and synaptic activity was recorded from identified neuronal cell types in slices of rat prefrontal cortex using patch clamp and simultaneous biocytin fillings. Results: Bath application of adenosine caused a hyperpolarisation of the membrane potential (V_m) of layer 5 pyramidal neurons (control -63.3 ±0.7 mV, 100 mM adenosine -66.8 ±0.8 mV, n=30, p<0.01) in a concentration dependent way (EC₅₀ 18.6 ±4.3 mM, n=8) while fast-spiking interneurons did not show a change in their V_m (control -73.1 ±1.8 mV, 100 mM adenosine -72.9 ±1.8 mV, n=6, p=0.11). However, the response of pyramidal neurons was highly variable, ranging from a maximum hyperpolarisation of 1.2 to 7.4 mV. Analysis of action potential and firing properties suggested that the size of the adenosine response was different for specific pyramidal cell subclasses: neurons with little spike adaptation showed a small adenosine response, while those with substantial adaptation showed a bigger response. In addition, the effect of adenosine was also layer specific: neither pyramidal neurons nor GABAergic interneurons in layer 2 showed a change in V_m upon adenosine application. Conclusions: The neuromodulator adenosine exerts its effects in a cell type, subclass and layer specific manner. In this way, adenosine modulates information processing at specific sites of the neuronal microcircuit. Unraveling adenosine's actions at the cellular and microcircuit level can eventually lead to an improved understanding of sleep homeostasis.