A distinct increase in cell firing activity is reported in prefrontal cortex during working memory tasks. The afferents that modulate this activity are not yet identified. Using in vivo intracellular recording and labelling of prefrontal cortical pyramidal neurons in anaesthetized rats, we systematically evaluated the influences of afferent projections arising from the ventral tegmental area (VTA) and mediodorsal thalamus (MD) by phasic electrical stimulation with a range of stimulus frequencies. Both VTA- and MD-responsive pyramidal neurons exhibited extensive intracortical axon arborization. Neither single shocks to the VTA at 0.2 Hz, nor low frequency trains of stimuli at 1–4 Hz (< 5 Hz) interrupted the periodicity of membrane bistability in bistable pyramidal neurons. However, high-frequency VTA-train stimulation (10–50 Hz) interrupted the bistability, and produced sustained membrane depolarizations accompanied by intense tonic firing in a frequency-dependent manner. Electrical stimulation of MD (10–50 Hz) did not produce sustained activity in the same PFC neurons. Thus, the sustained activity induced by high-frequency VTA trains is input selective. This effect of VTA-train stimulation was attenuated by systemic injection of the D1 receptor antagonist, SCH 23390, and blocked by acute dopamine (DA) depletion produced via α-methyl-para-tyrosine pre-treatment, suggesting that sustained cortical activity is mediated by DA. Chemical stimulation of VTA via intra-VTA infusion of NMDA induced sustained activity similar to VTA-train stimulation. Thus, while both VTA- and MD-responsive pyramidal neurons exhibited extensive intracortical axon arborization, VTA synapses (as opposed to MD synapses) may be critically positioned in the dendritic arborizations of anterior cingulate cortical pyramidal neurons, which may allow their modulation of sustained activity in prefrontal bistable neurons.