The earlier reported inhibition of rat liver nucleotide pyrophosphatase/phosphodiesterase I (EC 3.1.6.9/EC 3.1.4.1; NPP/PDE) by culture-grade acidic fibroblast growth factor (FGF-1) correlates with a low-Mr contaminant. 1H-NMR analyses revealed EDTA in the total-volume fractions of a gel-filtration experiment, where all the inhibitory activity of the FGF-1 preparation was recovered. NPP/PDE inhibition by EDTA (and by unfractionated FGF-1 or the EDTA-containing fractions) was time-dependent, blocked by the substrate p-nitrophenyl-dTMP, and strongly enhanced by glycine. The use of glycine buffers in earlier work was critical to the apparent inhibition by FGF-1. The results point to a conformational change favored by glycine that may be relevant to the biological role of NPP/PDE.