Two annexin I (anxI) genes, called cp35 and cp37, are expressed from the pigeon (Columba livia) genome, but they are regulated differently at both the transcriptional and post-transcriptional levels. The proximal promoter elements of these two genes are very similar. A conserved sequence from the cp35 and cp37 promoters bound specifically with proteins present in cropsac cell extracts. This sequence of DNA was used to screen a λgt11 cDNA expression library. Clones encoding two pigeon Y-box binding proteins (YB) were isolated. One of the pigeon YB cDNAs was found to be most similar to YB1 from other species, and the other was most similar to chicken YB2. Each YB is encoded by a single-copy gene in the pigeon, and their mRNAs are expressed in many tissues. On Northern blots, the sizes of the mRNAs encoding pigeon YB1 (pYB1) and pigeon YB2 (pYB2) were 1.8 and 1.7kb, respectively. The sequences of both pYB1 and pYB2 diverge from their previously identified relatives in the N-terminal domain `A'. Antisera were developed to unique peptide epitopes in YB1 or 2. Affinity-purified anti-YB1 and anti-YB2 detected immunoreactive proteins in extracts from a variety of pigeon tissues, including the cropsac. To confirm that pYB1 and pYB2 interact with the cp35 promoter, electrophoretic gel mobility shift reactions were carried out in the presence or absence of YB antibodies. Binding to the cp35 promoter was specifically neutralized by either anti-pYB1 or anti-pYB2. These results are the first evidence that two YB proteins simultaneously bind to a promoter element, and thereby may interact during regulation of gene expression.