This study investigated the effect of substituting grass silage (C3 photosynthetic plant product) with maize silage (C4 photosynthetic plant product) on the natural abundance carbon (δ13C) and nitrogen (δ15N) stable isotope composition of bovine muscle tissue. Forty-five continental crossbred heifers were assigned to one of three diets consisting of 3 kg of a barley-based concentrate plus grass silage, maize silage or an equal mixture (dry matter basis) of grass silage and maize silage, fed ad libitum, for 167 days. Substitution resulted in less negative δ13C values (P < 0.001) in lipid-free muscle and in lipid, and also a lower δ15N (P < 0.001) in lipid-free muscle. Feeding of maize silage was clearly reflected in the δ13C of muscle, with each 10% difference in the dietary C4 carbon intake resulting in a 0.9 to 1.0‰ shift of δ13C in lipid-free muscle and a 1.0 to 1.2‰ in lipid. Minimum detectable mean differences (95% confidence, power 0.80, n = 15) in this experiment were about 0.5‰ and 1.0‰ for δ13C of lipid-free muscle and lipid, respectively, and about 0.5‰ for δ15N of lipid-free muscle. The power analysis presented here is useful for estimating minimum isotopic differences that can be detected between any two groups of beef samples with a given number of replicates. It is concluded that carbon stable isotope ratio analysis of meat can be used to quantify C3/C4 dietary constituents in beef production. Copyright © 2005 John Wiley & Sons, Ltd.