A method is evaluated that employs variation in stable C and N isotopes from fractionations in C and N acquisition and growth to predict root biomasses of three plant species in mixtures. Moench (C4) were grown together in separate, three-species combinations. Surface roots (0-10cm depth) of each species from each of the two combinations were mixed in various proportions, and the relative abundances of lSN and 14N and 13C and 12C in prepared mixtures, surface roots of single species, and roots extracted from the 80-cm soil profile in which each species combination was grown were analyzed by mass spectrometry. An algebraic determination which employed the 613C, % 15N, and C and N concentrations of root subsamples of individual species accounted for more than 95% of the variance in biomass of each species in prepared mixtures with G. max, G. hirsutum, and S. bicolor. A similar analysis demonstrated species-specific differences in rooting patterns. Root biomasses of the C 4 monocots in each combination, S. scoparium and S. bicolor, were concentrated in the upper 20 cm of soil, while those of G. hirsutum and the woody P. glandulosa were largest in lower soil strata. Analyses of stable C and N isotopes can effectively be used to distinguish roots of species which differ in ratios of 15N to ~4N and ~3C to ~2C and thus to study belowground competition between or rooting patterns of associated species with different C and N isotope signatures. The method evaluated can be extended to quantify aboveground and belowground biomasses of component species in mixtures with isotopes of other elements or element concentrations that differ consistently among plants of interest.