Guanine is the major nitrogenous waste product in arachnids. It may serve as an indicator for allergenic mite faecal pellets. The present study assesses the correlation between the mite allergenicity of different house dust (HD) samples and their guanine content. Guanine content in HD samples was evaluated either by the Acarex-Test or determined quantitatively, especially for in-vitro methods. Using intradermal tests in selectively sensitized Dermatophagoides pteronyssinus (Dpt) patients, we evaluated the mite allergenicity of eight extracts obtained from HD samples with varying guanine contents. The weal area appeared to be proportional to the HD samples’ guanine content. A three-fold increase in skin reactivity was observed when the guanine content varied from 0.06% to more than 1%. Patients allergic to HD but not to Dpt had no significant reactions with HD extract prepared from a guanine-rich HD sample. For eighteen different HD samples coupled to paper discs, the percentage Dpt RAST binding values obtained with a Dpt-selectively sensitized patient serum pool was correlated with the guanine concentrations of HD samples (P < 0.01). The potencies of different HD extracts obtained from HD samples that varied in guanine content were also clearly distinguished on histamine-release titration curves. House dust obtained from homes at high altitude or from hospitals was generally guanine free. The HD sample guanine contents in homes where twelve Dpt-sensitized patients experienced symptoms were significantly higher than in homes where these patients were symptom free. The guanine colour test, a quick and easy technique, represents an important new development in indoor environmental investigations. It makes it possible to demonstrate mite faecal exposure and can be used to monitor the effectiveness of mite allergen avoidance measures.