Ten species of Culicoides were collected from 166 tree holes at 20 widely separated geographic locations to assess relationships with habitat pH. Wet tree holes (containing standing water) had a mean pH of 7.46 while dry tree holes (no standing water) had a mean pH of 8.60. Culicoides arboricola, C. guttipennis and C. villosipennis occurred in wet tree holes that had mean pH values of 7.66, 7.95 and 7.31, respectively. Dry tree holes, where C. hinmani, C. elemae, C. paraensis, C. nanus, C. snowi and C. footei occurred, had pH values ranging from 8.13 to 9.08. Culicoides lahillei, a dry tree hole species, was collected from habitats with a pH range similar to the wet tree hole species.