Transverse triple-quantum filtered NMR spectroscopy (TTQF) of 17O-water was used to study the properties of water in insulin solutions at different Zn2+ concentrations and pH values. It was established that strongly bound water molecules are already present in Zn-free insulin. On the assumption that the effective correlation time of a strongly bound water molecule, tau sb, is 10 ns, the apparent number of strongly bound water molecules was approximately 3 to 4 per insulin monomer. Addition of Zn2+ equivalent to approximately 2 g-atoms per hexamer did not produce substantial increases in the overall 17O-water TTQF signal intensity and apparent fraction of bound water. The dramatic enhancement of the TTQF signals observed for samples with a Zn2+/hexamer ratio greater than approximately 2:1 could be attributed to the increase in correlation time of the strongly bound water, due to the formation of higher-order oligomers of the protein.