The pituitary is a rich source of peptidylglycine alpha-amidating monooxygenase (PAM). This bifunctional protein contains peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) catalytic domains necessary for the two-step formation of alpha-amidated peptides from their peptidylglycine precursors. In addition to the four forms of PAM mRNA identified previously, three novel forms of PAM mRNA were identified by examining anterior and neurointermediate pituitary cDNA libraries. None of the PAM cDNAs found in pituitary cDNA libraries contained exon A, the 315-nucleotide (nt) segment situated between the PHM and PAL domains and present in rPAM-1 but absent from rPAM-2. Although mRNAs of the rPAM-3a and -3b type encode bifunctional PAM precursors, the proteins differ significantly. rPAM-3b lacks a 54-nt segment encoding an 18-amino acid peptide predicted to occur in the cytoplasmic domain of this integral membrane protein; rPAM-3a lacks a 204-nt segment including the transmembrane domain and encodes a soluble protein. rPAM-5 is identical to rPAM-1 through nt 1217 in the PHM domain; alternative splicing generates a novel 3‘-region encoding a COOH-terminal pentapeptide followed by 1.1 kb of 3‘-untranslated region. The soluble rPAM-5 protein lacks PAL, transmembrane, and cytoplasmic domains. These three forms of PAM mRNA can be generated by alternative splicing. The major forms of PAM mRNA in both lobes of the pituitary are rPAM-3b and rPAM-2. Despite the fact that anterior and neurointermediate pituitary contain a similar distribution of forms of PAM mRNA, the distribution of PAM proteins in the two lobes of the pituitary is quite different. Although integral membrane proteins similar to rPAM-2 and rPAM-3b are major components of anterior pituitary granules, the PAM proteins in the neurointermediate lobe have undergone more extensive endoproteolytic processing, and a 75-kDa protein containing both PHM and PAL domains predominates. The bifunctional PAM precursor undergoes tissue-specific endoproteolytic cleavage reminiscent of the processing of prohormones.