To standardize the procedure for isolating and culturing Lyme disease spirochetes, we modified the composition of the medium generally used for this purpose (BSK-II) and developed a system for its distribution. This medium contains no gelatin or agarose, and various components are used in proportions that differ from those in BSK-II. Each of the major proteinacious components was screened by substitution in samples of the complete product. The final medium was evaluated for the capacity to grow related spirochetes including Borrelia burgdorferi N40, Guilford, and JD-1 as well as strains of Borrelia hermsii (HS-1) and of Borrelia coriaceae (CO53). Each isolate developed from inocula containing as few as one to five organisms. Doubling time of B. burgdorferi during log-phase growth at 37 degrees C was 10 to 12 h. Lyme disease spirochetes were isolated in this medium from ear punch biopsies and dermal aspirates from naturally infected mice and rabbits, from dermal biopsies from a human patient, and by sampling field-collected deer ticks (Ixodes dammini). Cultured spirochetes remained infective to mice and to ticks. The medium can be stored at -20 degrees C or lower temperatures for at least 8 months without effect on its ability to support growth of small inocula to densities exceeding 10(8) spirochetes per ml. Lyme disease spirochetes remained infective to mice after being stored at -80 degrees C in this medium for at least 8 months. We anticipate that the availability of this standardized medium (Sigma Chemical Co.), supplemented with prescreened rabbit serum, will facilitate comparison of research results between laboratories and may eventually permit definitive clinical diagnosis of Lyme disease based on demonstration of the pathogen. The standardized medium is designated BSK-H.