The amino acid sequence of full-length ovine fibroblast growth factor-1 (FGF-1) was determined by a combination of protein and cDNA sequencing. FGF-1 cDNA analysis indicated that ovine kidney cells express mRNAs encoding both full-length FGF-1 and a truncated FGF-1 variant. An overall comparison of the ovine FGF-1 primary sequence to the eight species studied to date revealed a high degree of conservation, with ovine FGF-1 sharing 90 and 95% sequence identity with human FGF-1 and bovine FGF-1, respectively. Additionally, the FGF-1 proteins from the various species have conserved cysteine residues at positions 30 and 97 and contain acetylated amino-terminal alanine residues. Mass spectrometry analysis confirmed that the blocking group of ovine FGF-1 is also consistent with that of an acetyl-moiety. In contrast to the other FGF-1 proteins, the 154 residue primary sequence of ovine FGF-1 contains three unique amino acid differences: Arg9, Arg44, and Ile123. Ovine FGF-1, unlike human FGF-1, is a potent mitogenic factor for NIH 3T3 fibroblasts in the absence of heparin. In the presence of exogenous heparin, the mitogenic activity of ovine FGF-1 is potentiated slightly.