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Agave species are economically important plants in tropical and subtropical desert ecosystems as ornamentals as well as potential bioenergy crops. However, their relatively long life cycles and the current lack of biotechnology tools hinder their breeding. In this study, an efficient system for micropropagation was developed for Agave americana L. by using basal stems as explants and grown on a modified Murashige and Skoog medium (MSI) or a 1/2 MSI medium supplemented with various concentrations of 6-benzylaminopurine (BA) for shoot proliferation. The highest number of shoots (18.5 shoots/explant) from basal stems was obtained on MSI supplemented with 13.32 mu M BA. An efficient shoot regeneration system was also developed from leaf tissues. Combinations of auxin with cytokinin, basal media, and leaf regions were optimized for shoot induction. Adventitious shoot formation from leaf segments was induced and proliferated with combination ranging of 0.54 to 2.68 mu M [alpha-naphthaleneacetic acid (NAA)] with 8.88 to 13.32 mu M (BA), and the maximum frequency (approximate to 69%) was obtained with 2.68 mu M NAA plus 13.32 mu M BA. MSI medium and the basal segment of leaf affected shoot induction. The highest rooting frequency and mean number of shoots occurred in 1/2 MSI containing with 4.92 mu M indole-3-butyric acid (IBA) alone (90%, 3.4) or 1.48 mu M IBA plus 1.61 mu M NAA (92%, 5.2). Survival of in vitro plantlets after transfer and acclimatization to ex vitro conditions was 87%. This is the first complete protocol for micropropagation of A. anzericana.