In the present study, we have cloned a second insulin gene in zebrafish, and studied temporal and spatial expression of two zebrafish insulin genes. Zebrafish insulin a (insa) and insulin b (insb) mRNAs are derived from two different DNA contigs on chromosome 5 and chromosome 14 respectively, representing two different insulin genes. Real time PCR studies suggest that insa is a maternal and also a post-zygotic transcript. insa was observed at 1 hpf, and was rapidly degraded by 6 and 12 hpf, but was induced at 24 hpf (i.e. after pancreas formation). The expression levels at 24 hpf were approximately 220-fold higher than at 6 hpf and were significantly different from earlier time points. At 72 hpf (at the time of hatching), zebrafish insa mRNA levels tended to be higher than at 24 hpf and were approximately 727-fold higher compared to 6 hpf. This further increase in insa expression may be one of the many rapid physiological changes associated with hatching. insb expression was observed from1hpf and was significantly decreased from 12 hpf onwards. Its expression levels at 12 hpf and 24 hpf were approximately 2-fold, and 6-fold lower respectively, compared to its expression at 6 hpf. insb expression levels at 48 hpf were significantly lower than at 24 hpf, but not different from 72 hpf. Its expression levels at 72 hpf were approximately 61-fold lower than at 6 hpf. In situ hybridization studies showed insb expression in proliferating blastomeres at 3 hpf and 4 hpf. At later time points insb expression was restricted to the brain and pancreas (24 and 48 hpf). insa expression was observed in the pancreas at 24 and 48 hpf. The expression of insb in blastomeres and the head, suggest that insb could be acting as a pro-growth, survival and neuro-trophic factor during development. Pancreatic insa and insb may both be involved in the regulation of glucose homeostatsis as in mammals.