P19 embryonal carcinoma cells differentiate into neurons, astrocytes, and fibroblast-like cells following induction with retinoic acid. The mature neurons are capable of neurotransmitter release, and from functional synapses. We have previously shown that high culture density suppresses the cholinergic phenotype of P19 neurons. Here we demonstrate that increasing culture density accelerates the maturation of P19 neurons in a continuous manner. This is manifested by several criteria: increased efficiency of evoked [3H]aspartate release; decreased level of basal release; up-regulation of synaptic vesicle proteins; increased neurite outgrowth rate; and earlier segregation of axons and dendrites. While glutamate release is enhanced in dense cultures, the efficiency of [3H]GABA release is hardly affected, suggesting that P19 GABAergic neurons are not affected by culture density. The acceleration of neuronal maturation in dense cultures is also exhibited by the ability of dense, but not sparse cultures to release [3H]aspartate at an earlier day of differentiation. Furthermore, density effects are monitored already a few hours after plating the cultures, when neurite length in dense cultures is several fold higher than in sparse cultures. This indicates that commitment to a faster and coordinated maturation process occurs already very early during P19 neuronal differentiation.