Normal, preneoplastic, and neoplastic primary cultures of mouse mammary epithelial cells were distinguishable on the basis of water proton nuclear magnetic resonance (NMR) relaxation times--i.e., spin-lattice relaxation time (T1) and spin-spin relaxation time (T2). T1 values were 916 +/- 24 msec for normal cells, 1,029 +/- 24 msec for preneoplastic cells, and 1,155 +/- 42 msec for neoplastic cells. This method of distinction between normal and neoplastic cells (P less than 0.001) and normal and preneoplastic cells (P less than 0.005) supported previous findings in whole tissues. NMR relaxation times resulted in better distinction between these cell populations than any other technique except direct histology. The T1 and T2 values of water protons in cells grown in primary culture were higher than those of established mouse mammary cancer cell lines. The differences in T1 and T2 did not correlate with cellular hydration. The data suggested a basic difference in water-macromolecular surface interactions among normal, preneoplastic, and neoplastic cells.