During muscarinic modulation, principal neurons from layer V of rat medial entorhinal cortex (mEC) respond to repeated applications of a brief stimulus with a graded change in persistent firing frequency. This pattern of discharge has been proposed to represent an intrinsic mechanism for short-term memory operations. To investigate the implementation of persistent activity in mEC during development, we characterized the electrophysiological properties of layer V principal neurons in the mEC over a range of postnatal stages. We observed significant differences in both passive (resistance, time constant, and resting membrane potential) and active properties (threshold, action potential, and adaptation) of principal neurons from rats aged 5-7, 10-13, 16-19, and 21-23 days. We also examined the properties of muscarinic-dependent persistent activity in EC slices from different age groups. Recordings were conducted using the perforated-patch whole cell technique because persistent activity runs down in the ruptured-patch configuration. Although no neuron in the youngest group exhibited graded persistent activity in response to muscarinic receptor activation, this activity was recorded in the 10- to 13-day-old group and its occurrence increased from 69% in the 16- to 19-day-old group to 76% in the 21- to 23-day-old group. This postnatal increase in neurons endowed with persistent firing properties in mEC was found to parallel the increase in density of ChAT-positive immunostaining of fibers and the developmental changes in M1 muscarinic receptor mRNA levels. All these data suggest that the implementation of mnemonic properties in mEC principal neurons matches the ontogenic development of afferent cholinergic circuits and their signaling components.