Acidification of freshwaters via anthropogenic means is a global problem. Decreases in pH in these sys-tems have devastating effects on macroinvertebrate fauna especially Ephemeropterans. However, faunain naturally acidic waters with high concentrations of humic substances (HS) appear to be less affectedby acidification. This study investigated the ability of HS to decrease toxicity of low pH to mayflies(Atalophlebia spp.) in both hard and soft water environments. Laboratory trialling with both hard andsoft water was conducted to represent water chemistries commonly associated with anthropogenic andnaturally acidic freshwaters. Mayflies were exposed to decreases in pH between 7 (control) and 3.5 inthe presence of 0, 10 and 20 mg/L HS (Aldrich humic acid). HS increased mayfly survival by up to 45%in soft water and up to 39% in hard water. HS were also shown to increase 96 h LC50values in both soft(4.29, 0 mg/L; 3.99, 10 mg/L and 3.97, 20 mg/L) and hard water (4.58, 0 mg/L; 4.22, 10 mg/L and 4.07,20 mg/L). The results of this study are important in showing that HS can influence the toxicity of low pHenvironments; thus providing insight into why contrasting effects on biota are recorded from naturallyacidic compared with anthropogenically-acidified environments