Mammalian pituitary gland development is well established on a molecular and morphological level. In developmental biology the use of chick and zebrafish models is common but despite chicken pituitary gland development being characterised in 1952 these models have only recently been regularly employed in pituitary studies. The third member of the natriuretic peptide family, C-type natriuretic peptide (CNP), has been investigated in rodent embryos but little is known about its role in chick and zebrafish development. In our study we have used Gallus gallus and Danio rerio embryos to investigate the role of CNP in pituitary gland development. RNA in situ hybridisation probes were generated to detect expression of chick CNP3 gene and zebrafish CNP3 and Nppc-like (NPPCL) genes. The chick CNP3 probe was synthesised from a CNP3 EST (Univ. Manchester Chick EST Database). Primers were designed against zebrafish CNP and NPPCL. RT-PCR was carried out on whole mixed-age zebrafish cDNA and the PCR products purified and cloned using pGEM-T Easy vector. All plasmids were linearised, reverse transcribed and labelled with digoxigenin for detection with anti-digoxigenin antibody during in situ hybridisation. We have detected expression of CNP3 at days 4, 5 and 6 of Gallus gallus development (Hamburger Hamilton stages 2429), which was especially marked at day 4 in the developing chick pituitary gland. In Danio rerio CNP3 expression was detected from 48 h post fertilisation (hpf) in the brain and pituitary. NPPCL expression was widespread in zebrafish from 24 hpf but became more specific along the midline of the head up to 72 hpf, including expression in the pituitary. Our findings suggest CNP may be important in the crucial stages before and after Rathkes pouch formation in the chick. In zebrafish CNP appears to be important after internalisation of the pituitary and during the later stages of pituitary patterning.