The insulin release in response to glucose was studied in perifused isletlike cell clusters (ICCs) obtained from human fetal or neonatal pancreases at various stages of development: 12-15 gestational wk (n = 7), 17-20 wk (n = 13), 22.5 wk (n = 2, 1 diabetic pregnancy), and 26-44 wk (n = 6, postnatal samples). The ICCs were stimulated with 20 mM glucose and subsequently with 10 mM theophylline plus 20 mM glucose as a viability test. Insulin release increased to a detectable level (greater than 0.1 pg.ICC-1.min-1) during glucose stimulation in four of seven of the youngest fetuses. At 17-20 wk the basal rate of insulin release had increased by at least 15-fold above the detection limit (1.5 pg.ICC-1.min-1), and glucose promoted a sustained monophasic response that was on the average 1.6-fold higher than the basal level. The response was significant (P less than .05) in 9 of 13 experiments. With postnatal ICC (gestational age 26-44 wk), an early-phase peak response was observed in 5 of 6 experiments. The mean rates of insulin release after 5-12 min of glucose stimulation were 4.8 pg.ICC-1.min-1 in newborn infants and 2.1 pg.ICC-1.min-1 in 17- to 20-wk fetuses. The corresponding mean relative insulin responses (stimulated to basal) were 3.3-fold (range 1.1-7.5) and 1.6-fold (1.0-3.4), respectively (P less than .05, Mann-Whitney U test). The results suggest that the human fetal pancreas is already responsive to glucose during the first half of gestation, but the biphasic insulin release does not start to mature until the postnatal phase.