The comparison of N-15 natural abundance (delta N-15) between fixing and non-fixing reference species, is the most feasible method for nitrogen fixation studies in xeric shrubs. However, it assumes that both species use the same sources of soil N, the delta N-15 of such source is clearly different from zero, and the difference between the delta N-15 of both species is >= 5 parts per thousand. On the other hand, the analysis of the difference between the delta N-15 of nodules and leaves (Delta delta N-15(n-1)) does not require a reference species. To evaluate the effect of water availability on nitrogen fixation, we analyzed the variation of delta N-15 and Delta delta N-15(n-1) in watered and unwatered plants of Adesmia volckmanni Phil. (Fabaceae) and Mulinum spinosum (Cav.) Pers. (Apiaceae), formerly proposed as non-fixing reference species. However, we found that M. spinosum was not valid as reference species to study N fixation in Adesmia volckmann because its delta N-15 was closer to zero than that of the presumably fixing species, suggesting they use different soil N sources. In addition, water availability did not affect biological fixation after watering because both leaf delta N-15 and Delta delta N-15(n-1) of A. volckmanni did not change either as soil dried up along the growing season nor in response to watering. However, the presence of nodules itself, the delta N-15 closer to zero than in a former dry year, and the Delta delta N-15(n-1)(+ 1.23 parts per thousand) suggest that biological fixation could have occurred before watering, probably during early spring. (C) 2014 Elsevier Ltd. All rights reserved.