Cultured keratinocytes are used for wound coverage in some patients with extensive burns, but there are difficulties in assessing when the keratinocytes are ready for grafting. The morphologic characteristics of developing cultured skin provide a method of identifying mature grafts. This study investigated the structure of cultured keratinocytes (apical and basal cells) by means of Scanning Electron Microscopy. Both primary cultures and secondary-passage cultured keratinocytes were studied from subconfluence through maturity and into senescence. At subconfluence, the keratinocytes comprised one cell layer, contiguous in some areas, and appeared as irregular, polygonal cells with protruding nuclei. Contiguous cell membranes had microvillae that varied in pattern and length. With initial stratification (two layers), the apical cells were immature and had finger-like microvillae that projected from rounded cells. The basal cells were uniformly covered with short microvillae. Additional stratification (four to five cell layers) gave rise to an apical layer that resembled a quasi-stratum corneum in which cells lacked both cytoplasm and nuclei. Basal cells were plump and regular, with microvillae that protruded from intact cell membranes. These characteristics can be used as indicators of mature keratinocytes to optimize the take of keratinocyte grafts. At senescence, many basal cells were flattened and lacked part of their cell membrane. There are defects in the cultured keratinocyte cell sheet that would limit graft take. A comparison between primary culture and secondary passage noted that primary-culture cultured keratinocytes were less suitable for grafting purposes because of the variation in maturation rates between dishes that were plated at the same time. This study determined that morphologically optimal grafting occurs during secondary passage of cultured keratinocytes, approximately 3 to 5 days after confluence is reached.