The present study, using proton nuclear magnetic resonance relaxation(H1 NMR) measurements, was undertaken to quantitate water fractions with different mobility in the brain tissue obtained from New Zealand White rabbit pups. Serial studies were carried out at the postnatal age of 0-1, 24, 48, 72, and 96 h in pups nursed with their mothers and suckling ad libitum (group I) and in those pups separated from their mothers and completely withheld from suckling (group II). Tissue water content(desiccation method) and T1 and T2 relaxation times (H1 NMR method) were measured. Free, loosely bound, and tightly bound water fractions were calculated by applying multicomponent fits of the T2 relaxation curves. It was demonstrated that brain water content and T1 and T2 relaxation times did not change with age in the suckling pups. In pups withheld from suckling brain water decreased from 89.4 ± 0.5% at birth to 87.7 ± 0.1% at the age of 96 h (p < 0.05), T1 remained unchanged, and there was a significant fall in T2 by the age of 72 h (188 ± 12 versus 178 ± 4 ms,p < 0.05) and 96 h (171 ± 6 ms, p < 0.01). Partition of brain water into bound and free fractions as derived from biexponential fits of T2 decay curve showed that the percent contribution of bound water fraction in pups of group I fell progressively from 61% at birth to 3% at the age of 72-96 h (p < 0.05). This fall was accelerated by the complete deprival of fluid intake, and the level of about 4% could be attained as early as the age of 24 h. Triexponential analysis of T2 relaxation curves revealed that the loosely bound fraction (middle component) predominated over the free (slow component) and the tightly bound (fast component) water fractions. In response to withholding fluid intake, the free water fraction increased 4-fold at the expense of tightly bound brain water. It is concluded that the majority of neonatal brain water is motion-constrained. The free, the loosely bound, and the tightly bound water fractions appear to be interrelated; from the brain water store water can be released to supply free water for volume regulation.