Background: House dust mites (HDM) such as Dermatophagoides pteronyssinus and Dermatophagoides farinae represent a major cause of type 1 allergies worldwide. Hence large quantities of well-characterized HDM extracts are needed to prepare pharmaceutical-grade allergy vaccines. To this aim, the present study was undertaken to define optimal conditions for large-scale cultures. Methods:D. pteronyssinus and D. farinae were grown on different media combining various proportions of wheat germ, yeast and synthetic amino acids (the latter resembling the composition of the human stratum corneum). Extracts thus obtained were analyzed for their total allergenic activity, as well as major allergen and protein contents, using immunosorbent assays, HPLC, immunoblotting, two-dimensional electrophoresis and peptide mass fingerprinting. Results: An optimal culture medium (Stalmite APF®) based on wheat germ, yeast and amino acids in defined proportion (42, 42 and 15% w/w, respectively) was selected to grow various HDM species with high yields. A detailed proteomic analysis revealed that D. pteronyssinus extracts generated under such conditions did not contain allergens originating from culture medium components and that major prevalent HDM allergens (i.e. groups 1, 2, 7, 10, 13 and 20) are found among the most abundant proteins in the D. pteronyssinus extract. Semiquantitative dot-blot assays confirmed the presence of Der p 3–10 as well as Der p 13 and 14 allergens within the extracts. Conclusions: We developed a well-defined medium allowing to grow various HDM species at an industrial scale in a highly reproducible manner. Extracts from mites produced under such pharmaceutical conditions contain all the relevant allergens for desensitization purposes and in vivodiagnosis.