Fifty-four analogs of human growth hormone-releasing factor (hGRF) substituted with a single D-amino acid were synthesized by solid phase methodology. Their capacity to release growth hormone was tested on rat anterior pituitary cells in monolayer culture. Among the series of 28 analogs, which had the amino acid at each position of hGRF (1-29)NH2, except glycine at position 15, substituted by the corresponding D-isomer, [D-Ala2]-, [D-Asp3]-, [D-Asn8]-, [D-Tyr10]-, [D-Asp25]-, [D-Met27]-, [D-Ser28]-, and [D-Arg29]hGRF(1-29)NH2 were as potent as hGRF(1-29)NH2, while [D-Ile5]-, [D-Phe6]-, [D-Thr7]-, and [D-Val13]hGRF(1-29)NH2 showed quite low potencies. Effects of substitution with other D-amino acids in positions 2,3,8,9,10 and 11 were also studied. In most cases, the resulting analogs showed decreased potency, but still retained high intrinsic activity. Only [D-Arg2]hGRF(1-29)NH2 showed very low intrinsic activity and some antagonistic property.