Isolated cellular nidi stem cells from the anterior and posterior midgut of larvae of the red turpentine beetle, Dendroctonus valens LeConte, proliferate and differentiate in vitro when cultured in RPMI 1640 medium, in the presence of 20-hydroxyecdysone, fat body extract from Zophobas morio (Blanchard) (Coleoptera: Tenebrionidae) pupae, and conditioned medium. Our data suggest that the stem cells did not increase exponentially as in typical cultures. However, the early presence of stem cells in larval cultures indicates that the proliferation process is higher than in cultures from adult D. valens. After 2 wk in culture, up to 10% of cells were observed in various stages of differentiation. The mean duration of midgut cultures was 82 d.