The probability of transmitter release at synaptic terminals is one of the key characteristics of communication between nerve cells because it determines both the strength and dynamic properties of synaptic connections. To assess the distribution of the release probabilities at excitatory synapses on supragranular pyramidal cells in rat visual cortex, we have used the MK-801, a blocker of the open N-methyl-d-aspartate (NMDA) receptor-gated channels. With this method, the release probability can be calculated from the time course of the blockade of NMDA-receptor mediated postsynaptic currents in the presence of MK-801. At temperatures >32 degrees C, the distribution of release probabilities covered the range from 0.05 to 0.43 [mean: 0.171 +/- 0.012 (SE), n = 65], being skewed toward low values. When estimated at room temperature (22-25 degrees C), the release probabilities were significantly lower (mean: 0.123 +/- 0.009, n = 54), and almost the whole distribution was restricted to values <0.2. Furthermore, warming from room temperature to >32 degrees C led to a pronounced overshooting increase of the release probability. Taken together, the results of the present study show that release probabilities at synapses formed onto layer 2/3 pyramidal cells in the visual cortex vary significantly, but values >0.3 are rare and the results obtained either at room or variable temperature differ significantly from those made under conditions of constant temperature in the physiological range.