We have prepared Lactobacillus casei dihydrofolate reductase containing biosynthetically incorporated (2S,4S)-5-fluoroleucine ([5-F]-Leu DHFR) and have obtained its H-1 and F-19 NMR spectra at 9.4 Tesla. The F-19 spectrum of [5-F]-Leu DHFR showed 12 fairly sharp peaks (one containing two overlapped signals) for the 13 leucine residues in DHFR, covering a chemical shift range of 15 ppm. The large range of chemical shifts observed could not be explained solely in terms of the electrostatic field effects due to local charge fields and is thought to have a second contribution from side-chain conformational differences (gamma-gauche effects) between different leucine residues, making F-19 NMR of aliphatic amino acids in proteins a potentially useful new probe of protein structure.