Three rat 13762NF mammary adenocarcinoma clones and cell lines of different metastatic potentials (MTLn3, MTC, and MTPa) were studied for their proton nuclear magnetic resonance spectral characteristics as intact cells in vitro and after chloroform/methanol, neuraminidase, or ethanol treatments. The intact-cell spectral characteristics of the highly metastatic tumor cell clone MTLn3 were clearly distinguished from the less metastatic clone MTC or the parental MTPa cell line on the basis of spectral peaks in the range of 0"9 to 1-45 p.p.m, broad peaks near 2"0 p.p.m., and peaks in the range of 2"75 to 3"2 p.p.m. Glycoproteins are among the molecules known to have resonances in these upfield spectral regions, and these tumor cell subpopulations have previously been shown to possess characteristic quantitative differences in cell surface, metastasis-associated glycoproteins. Treatment of the cells with neuraminidase or ethanol, or extraction with chloroform/methanol increased spectral detail and also revealed characteristic differences in spectral peaks between the tumor cell subpopulations. The identity of the cellular components responsible for these spectral characteristics are unknown, but some clearly arise from differences in the extractable lipids present in the tumor cell suhpopulations. Further study will be required to determine if the spectral differences described in this preliminary report are directly related to the known biochemical characteristics of the highly metastatic clone, and if the observations have general relevance to metastatic potential or are a singular feature of these cells. However, these initial results suggest that manipulation of factors which allow unmasking of spectral detail combined with the use of prescribed tumor cell subpopulations may aid in using proton NMR to identify and define biochemical or structural differences related to the metastatic potential of tumor cells.