Analogs of the 29-amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) or Lys-NH2 in position 29 have been synthesized by the solid-phase method, purified, and tested in vitro. Except for one peptide, all analogs contained desaminotyrosine (Dat) in position 1. All contained Nle27 in order to avoid oxidation of Met27. Some peptides contained one or more additional L- or D-amino acid substitutions in positions 2, 12, 15, 21, 27 and/or 28. Analogs [Dat1, Ala15, Nle27, Asn28]GH-RH(1-28)Agm (II, [Asn28]-Mz-2-51); [Dat1, Ala15, D-Lys21, Nle27, Asn28]GH-RH(1-28)Agm (III, MZ-3-125); and [Dat1, D-Asn8, Ala15, D-Lys21, Nl27, Asn28]GH-RH(1-28)Agm(IV, MZ-3-129) were 5.7, 2.8, and 3.9 times more potent in vitro, respectively, than GH-RH(1-29)NH2. However, if we compare the potencies of peptides II and III (analogs of the bovine sequence) with those of the analogs of human GH-RH (XII and XIII) [Dat1, Ala15, Nle27]GH-RH(1-28)Agm; [Dat1, Ala15, D-Lys21, Nle27]GH-RH(1-28)Agm, respectively, the GH-releasing potency was decreased by 50% and 33%, respectively, by the incorporation of Asn28. Our studies indicate that Lys-NH2 at the C-terminus of GH-RH(1-29) and/or beta-Ala, GABA (gamma-aminobutyric acid), and Phe in position 15 are disadvantageous, but potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.