Ammonia is an important air and water pollutant, but the spatial variation in its concentrations presents technical difficulties in accurate determination of ammonia emissions from animal feeding operations. The objectives of this study were to investigate the relationship between ammonia volatilization and delta15N of dairy manure and the feasibility of estimating ammonia losses from a dairy facility using chemical markers. In Exp. 1, the N/P ratio in manure decreased by 30% in 14 d as cumulative ammonia losses increased exponentially. Delta 15N of manure increased throughout the course of the experiment and delta15N of emitted ammonia increased (p<0.001) quadratically from -31 per thousand to -15 per thousand. The relationship between cumulative ammonia losses and delta15N of manure was highly significant (p<0.001; r2=0.76). In Exp. 2, using a mass balance approach, approximately half of the N excreted by dairy cows (Bos taurus) could not be accounted for in 24 h. Using N/P and N/K ratios in fresh and 24-h manure, an estimated 0.55 and 0.34 (respectively) of the N excreted with feces and urine could not be accounted for. This study demonstrated that chemical markers (P, K) can be successfully used to estimate ammonia losses from cattle manure. The relationship between manure delta15N and cumulative ammonia loss may also be useful for estimating ammonia losses. Although promising, the latter approach needs to be further studied and verified in various experimental conditions and in the field.