Here, we describe a new protocol for the induction of direct somatic embryogenesis of Agave fourcroydes through thin cell layer (TCL) culture technology. The protocol was optimized for the main factors known to affect the process, including the type of explant (stem or leaf tissue), type and concentration of exogenous growth regulators (alpha-naphthalene acetic acid [NAA], 2,4-diclorophenoxyacetic acid [2,4-D], 4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid [picloram], and 3,6-dichloro-2-methoxybenzoic acid [dicamba]), and the influence of plant genotype. Thin tissue segments cut transversally (tTCLs) from stems of in vitro-cultured plants gave the best embryogenic response when cultured with 2.26 mu M dicamba (92.22 embryos/explant) or 2.07 mu M picloram (81.72 embryos/explant). It was interesting to observe that the embryogenic capacity of these tissues was affected by the presence of 6-benzylaminopurine (BA) in the culture medium in which the explant donor plantlets were maintained. Thirteen clonal lines (each derived from a different parental plant), compared for their embryogenic competence under the same culture conditions, produced very different embryogenic responses that varied from very high (117 embryos/explant) to null. The histological analysis revealed that the amount of meristematic tissue present in the tTCLs varied according to the region of the stem (apical, middle, or basal) from which they originated. The cells of the vascular procambium became competent and developed into cell lines that formed embryos, either by a unicellular or a multicellular pathway. Mature embryos germinated in half-strength Murashige and Skoog medium without growth regulators and 85% of regenerated plants was successfully acclimatized in a greenhouse.