The goal of this research was to induce neuron-like properties in Sf21 cells, an insect ovarian cell line, which could lead to a new high-throughput insecticide screening method and a way to mass produce insect neuronal material for basic research. This study applied differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, a maximum of ca. 30 % of Sf21 cells expressed an apparent neuronal morphology of unipolar, bipolar, or multipolar axon-like processes within 2-3 days. Maximal differentiation occurred after 2 days in the presence of 50 μM 20-HE or 3 days in 10 μM insulin. Both 20-HE and insulin displayed time- and concentration-dependent differentiation with biphasic curves, suggesting that two binding sites or processes were contributing to the observed effects. In addition, combinations of 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system stimulant, inhibited induction of elongated processes by 20-HE and/or insulin, with an IC(50) of 9 nM for 20-HE, and the inhibition was incomplete, resulting in about one-quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies the studies of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined.