Thyrotropin-releasing hormone prohormone contains multiple copies of TRH linked together by connecting sequences. Like other plurifunctional prohormone proteins, pro-TRH undergoes differential proteolytic processing in various tissues to generate, beside authentic TRH, several other novel peptides corresponding to C-terminally extended forms of TRH and connecting fragments. The pro-TRH connecting peptides are, together with TRH, predominant storage forms of TRH-precursor related peptides in the rat hypothalamus. Connecting peptides are co-localized with TRH in the median eminence nerve endings and co-released through a mechanism involving voltage-operated Ca2+ channels. The connecting peptide Ps4 is involved in potentiation of the action of TRH on thyrotropin hormone release by pituitary in vitro and in vivo through interactions with a specific pituitary cell receptor coupled to dihydropyridine and omega-connotoxin sensitive Ca2+ channels of the L-type. It also causes dose-dependent increases in the steady state levels of mRNAs of TSH and prolactin through stimulation of the respective gene promoter activities. These findings indicate that Ps4 and TRH, two peptides which originate from a single multifunctional biosynthetic precursor, can function on the same target tissues in a coordinate manner to promote hormonal secretion. This suggests that differential processing of the TRH prohormone may have the potential to modulate the biological activities of TRH.