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Highlights • 15N-enriched samples of corn and sunflower were used. • 15N abundance was lower in ADFom than in aNDFom and total nitrogen. • Current ADIN analysis overestimates the N associated to ADFom (ADIN fraction). • ADIN oversestimation was greater in fibrous samples, but decreased with high lignification. • Using nitrogen-free reagents in the ADFom analysis is suggested. Abstract The acid detergent insoluble nitrogen (ADIN) fraction of feeds is usually assumed to be indigestible, and some feeding systems for ruminants use the ADIN analysis as a measure of nitrogen (N) availability. In a preliminary study conducted with 3 different feeds, we observed lower 15N abundance in ADIN compared with that in both total N and neutral detergent insoluble N (NDIN). Based on these results, we hypothesized that ADIN analysis overestimates the amount of N associated to acid detergent fibre (ADFom) due to N-contamination from cetyltrimethylammonium bromide (CTAB), a reagent used in the ADFom analysis. The objective of this work was to verify lower 15N abundances of ADIN than of total N and NDIN in different feeds, and to assess the magnitude of ADIN contamination. Samples from one corn crop (aerial biomass, grain and root) and two sunflower crops (SF; aerial biomass and seeds) were analysed for 15N abundance and content of total N, NDIN and ADIN. Samples had been 15N-labelled by fertilizing with either 15NH 4 NO 3 or NH 4 15NO 3 , and there were three replicates per type of sample. The 15N-abundance of total N and NDIN was similar (P > 0.05) for all samples, whereas that of ADIN was lower (P < 0.001 to 0.037) than values for total N and NDIN for all samples, excepting for SF seeds from one crop. Estimates of N-contamination of ADIN from CTAB were not affected by the fertilizer used to label N (P ≥ 0.207), and values (g/g) ranged from 0.129 for corn grains to 0.447 for corn aerial biomass. N-contamination of ADIN was positively correlated with neutral detergent fibre (aNDFom), ADFom, hemicelluloses and cellulose content (P < 0.001; r = 0.770 to 0.905; n = 18 and 22 for corn and SF, respectively), and negatively (P < 0.001 to 0.048) with ADIN content (expressed as proportion of ADFom). The negative correlations (P < 0.001) between N-contamination of ADIN and lignin (Lignin (sa))/ADFom ratio observed for all samples would indicate that N-contamination of ADIN was reduced by cell wall lignification. In conclusion, the results showed that the use of CTAB in the ADFom analysis resulted in increased ADIN values, and therefore overestimated the amount of N associated to ADFom, questioning the use of ADIN as a parameter indicative of N availability. The possibility of replacing CTAB by a N-free reagent in the ADFom analysis should be explored. [ABSTRACT FROM AUTHOR]