Micropropagated `Festival' red raspberry (Rubus idaeus L.) shoots were rooted in specially constructed plexiglass chambers in ambient (340 ± 20 ppm) or enriched (1500 ±50 ppm) CO2 conditions on a medium containing 0, 10, 20, or 30 g sucrose/liter. Plantlet growth and leaf 14CO2 fixation rates were evaluated before and 4 weeks after ex vitro transplantation. In vitro CO2 enrichment promoted in vitro hardening; it increased root count and length, plantlet fresh weight, and photosynthetic capacity but did not affect other variables such as plantlet height, dry weight, or leaf count and area. No residual effects of in vitro CO2 enrichment were observed on 4-week-old transplants. Sucrose in the medium promoted plantlet growth but depressed photosynthesis and reduced in vitro hardening. Photoautotrophic plantlets were obtained on sucrose-free rooting medium under ambient and enriched CO2 conditions and they performed better ex vitro than mixotrophi plantlets grown with sucrose. Root hairs were more abundant and longer on root tips of photoautotrophic plantlets than on mixotrophic plantlets. The maximum CO2 uptake rate of plantlet leaves was 52% that of greenhouse control plant leaves. This did not change in the persistent leaves up to 4 weeks after ex vitro transplantation. The photosynthetic ability of persistent and new leaves of 4-week-old ex vitro transplants related neither to in vitro CO2 nor medium sucrose concentration. Consecutive new leaves of transplants took up more CO2 than persistent leaves. The third new leaf of transplants had photosynthetic rates up to 90% that of greenhouse control plant leaves. These results indicate that in vitro CO2 enrichment was beneficial to in vitro hardening and that sucrose may be reduced substantially or eliminated from red raspberry rooting medium when CO2 enrichment is used.