A cDNA library was prepared from mRNA isolated from anterior pituitary glands of incubating bantam hens, in which prolactin mRNA levels were predicted to be very high. Nine clones, representing abundant mRNA species, were identified and shown to contain homologous sequences. Two clones, of 871 bp and 580 bp, were analysed by DNA sequencing. The shorter clone was found to be a truncated cDNA product but otherwise identical to the longer clone. The 871 bp cDNA, PRL101, contains an open reading frame capable of encoding a polypeptide of 229 amino acids. This putative polypeptide has a high degree of homology to mammalian prolactins (approximately 70%), strongly suggesting that PRL101 encodes chicken preprolactin. The protein was predicted to have a 30 amino acid signal sequence which would be cleaved off to give a mature protein of 199 amino acids. The peptide sequence also had a 26% homology to chicken growth hormone, which is related to prolactin. This similarity confirms the conclusion that PRL101 is a chicken prolactin cDNA clone. An abundant mRNA of approximately 880 b was detected in poly(A)+ RNA from pituitary glands probed with PRL101. Analysis of chicken genomic DNA showed that there is one copy of the prolactin gene in the genome. PRL101 hybridized strongly to genomic DNA from closely related galliforms (quail and turkey) and less strongly to DNA from more distantly related species (duck and ring dove).