The most recently discovered member of the family of natriuretic peptides, C-type natriuretic peptide (CNP), exerts many pharmacologic actions similar to its structural homolog A-type natriuretic peptide (ANP). Like ANP it failed to significantly alter prolactin release from dispersed, rat anterior pituitary cells incubated under static or dynamic conditions. Unlike ANP, however, which inhibits prolactin secretion in vivo by a hypothalamic action, CNP injection into the third cerebroventricle significantly stimulated prolactin secretion in ovariectomized, conscious rats. The effect was highly significant 15 min after injection and transient, lasting 30 min in animals injected with 2 nmole CNP. In a companion group of rats, significant inhibition of plasma prolactin levels was observed after central administration of similar doses of ANP. These results suggest differing hypothalamic actions of the CNP and ANP perhaps mediated by multiple natriuretic peptide receptors present in the tissue. Further, they provide additional support for unique roles exerted within the central nervous system by these structural homologs.