Homozygous 40- to 90-day-old "little" mice (lit/lit), derived from the C57BL/6J strain, have been used to set up an in vivo body weight gain bioassay for GH whose performance has been compared to the widely used hypophysectomized rat assay. A log dose-response curve has been analyzed in order to choose doses in the linear range that are suitable for setting up useful, precise, and economical 2 x 2 factorial assays. A comparison between the response in the two sexes has also been carried out, showing no significant difference between male and female little mice of this age. Three assays have been carried out in these animals for the potency determination of a local human GH (hGH) reference preparation in terms of First International Standard of GH (human) for bioassay (WHO 80/505), comparing them to a classical assay in hypophysectomized rats performed with the same preparations. The calculated potency values were in good agreement, while the statistical parameters indicated a comparable assay precision, even in a practical and rapid 4-day assay. We suggest the substitution of hypophysectomized rats with little mice for this in vivo test, still required for potency and bioidentity determination of clinical preparations of recombinant hGH. This avoids the highly invasive, costly, and time-consuming surgery, providing a faster, more flexible, and economical assay method, while still directly measuring parameters of in vivo linear growth in an animal model closely related to isolated hGH deficiency type I.