Insulin has metabolic, growth, and differentiation effects in chicken embryos in vivo and it is required for normal development. Whether the pancreas is the sole source of insulin in embryogenesis is controversial. In the present study we investigated (1) the developmental pattern of expression of the chicken insulin gene in the pancreas; (2) the expression of the insulin gene in three nonpancreatic tissues, liver, brain, and lower limb, during chicken development; and (3) the expression of the insulin gene at prepancreatic stages and during chicken embryo organogenesis. Hybridization of synthetic species-specific insulin oligonucleotides to pancreatic frozen section in situ and to Northern blots revealed a major increase in insulin messenger RNA (mRNA) levels during the third week of embryonic development. The hybridization histochemistry showed both an increase in the levels of insulin mRNA per pancreatic islet and, in addition, an increase in the number of insulin mRNA containing islets with development. By Northern analysis there was a major polyadenylated transcript of 0.6 kb, which increased in abundance approximately 30-fold during this interval. Under the same stringency conditions used for pancreatic RNA an insulin transcript was detected in liver RNA blots. The abundance of this hepatic insulin mRNA was about 100-fold less than the pancreatic insulin mRNA and, in contrast to the latter, did not increase in late development. Primer extension experiments demonstrated that the insulin transcripts of pancreas and liver had similar 5' ends. No insulin mRNA was detected by Northern analysis or primer extension either in whole brain or lower limb total RNA from several developmental stages. A very low abundance insulin mRNA was detected in whole embryo at Day 8 and body regions at Day 4 and Day 5 when organogenesis of the pancreas takes place. Interestingly, a polyadenylated insulin transcript was detected, as well, in whole Day 2 and Day 3 embryos (stages 10 to 20, with 20 to 40 somites) before differentiation of beta cells occurs. Thus, there is differential developmental regulation of the insulin gene in several chicken embryo tissues and the expression of insulin precedes pancreatic maturation. These findings support the proposed role of insulin in differentiation and development in vivo and suggest a paracrine type of action of the hormone in early embryos before blood circulation begins.