The development of micropropagated banana plants during the in vitro growth phase prior to acclimatization was studied both in tight vessels under two different photosynthetic photon flux densities (PPFD of 30 and 240 μmol m −2 s −1) and in continuously flushed vessels under three atmospheric CO 2 concentrations (0.034, 0.24 and 4.0%) at 240 μmol m −2 s −1 PPFD. In tight vessels at low PPFD, the CO 2 originating from dark respiration was partially fixed during the light period, indicating photosynthetic activity by the plants in vitro. At high PPFD, CO 2 originating from dark respiration was rapidly fixed in the early hours of the light period and CO 2 concentration became the limiting factor for photosynthetic activity. Plants in vitro grown under high PPFD accumulated 2.3 times the dry matter achieved by plants in low PPFD. However, this developmental advantage acquired in vitro was not maintained ex vitro at the end of the acclimatization phase (on a leaf area basis). In continuously flushed vessels, treatments with 0.24% and 4.0% CO 2-enriched atmospheres enhanced dry matter accumulation in vitro by 1.6 and 2.3 times, respectively, as compared to a 0.034% CO 2 treatment. Twenty days after transfer ex vitro, the development of plants (on a leaf area basis) from these CO 2 treatments was no longer significantly different. The relative growth rate ex vitro was lower for plants cultured in a CO 2-enriched atmosphere in vitro than for those cultured at 0.034% CO 2. Plants cultured in vitro in continuously flushed vessels, after 8 weeks ex vitro, produced as much as five times the foliar area of plants grown in vitro in tight vessels.