In gnathostomes, the adenohypophysis, a component of the hypothalamo-hypophysial complex, is believed to develop through hierarchically organized epigenetic interactions based primarily on the topographical relationships between tissues. From a comparison of developmental processes and gene expression patterns of pituitary-related genes between the agnathan species, lampreys and gnathostomes, we speculate on the evolutionary pathway of the vertebrate adenohypophysis. In the lamprey, this is derived from the nasohypophysial placode (NHP) that develops anterior to the oral ectoderm. The NHP can be identified by the expression of LjPitxA, before actual histogenesis, but it is initially distant from the future hypothalamic region. Subsequently, the NHP expresses both LjFgf8/17 and LjBmp2/4a gene transcripts, and grows caudally to establish a de novo contact with the hypothalamic region by the mid-pharyngula stage. Later, the NHP gives rise to both the adenohypophysis and an unpaired nasal organ. Thus, the topographical relationship between the NHP and the hypothalamic region is established secondarily in the lamprey, unlike gnathostomes in which the equivalent relationship appears early in development. Comparing the developmental pattern of the amphioxus homologue of the adenohypophysis, we hypothesize that a modification of the regulation of the growth factor encoding gene lies behind the evolutionary changes recognized as heterochrony and heterotopy, which leads to the gnathostome hypophysial developmental pattern. J. Exp. Zool. (Mol. Dev. Evol.) 300B:32–47, 2003. © 2003 Wiley-Liss, Inc.