Human keratinocytes derived from explants of cheek (buccal) mucosa grow vigorously in culture and can be subcultivated twice. The structure of the oral keratinocytes in vitro is the same in primary cultures and subcultures. The cells stratify, are characterized by well-developed tonofibrillar-desmosomal complexes, and rarely exhibit signs of terminal differentiation. Unique features of the culture system that favor keratinocyte growth are: incubation at 34 degrees C, inclusion of 0.5% dimethyl sulfoxide in the culture medium, and initiating subcultures as 5.0 mm colonies containing 100,000/20 microliter of medium. One primary culture can yield 6 first-passage subcultures, which subsequently achieve confluence in 10-12 days. Such cultures are a useful source of human keratinocytes that stratify but generally do not undergo terminal differentiation.