1. Ketamine is a potent bronchodilator which relaxes airway smooth muscle (ASM). Clinically, ketamine is used as a 1:1 racemic mixture of enantiomers that differ in their analgesic and anaesthetic effects. The aim of this study was to determine whether there was a difference between the enantiomers in their ability to relax isolated ASM and to explore mechanisms responsible for any observed differences. 2. Canine tracheal smooth muscle strips were loaded with fura-2 and mounted in a photometric system to measure simultaneously force and [Ca2+](i). Calcium influx was estimated by use of a manganese quenching technique. 3. In strips stimulated with 0.1 μM ACh (EC50) R(-)-ketamine (1-100 μM) caused a significantly greater concentration-dependent decrease in force (P < 0.0001) and [Ca2+](i) than S(+)-ketamine (1-100 μM) (P < 0.0005). In contrast, there was no significant difference between the enantiomers in their ability to inhibit calcium influx (45% decrease in influx rate for R(-)-ketamine and 44% for S(+)-ketamine, P = 0.782). In strips contracted with 24 mM isotonic KCl (which activates voltage-operated calcium channels), the enantiomers modestly decreased force and [Ca2+](i) there was no significant difference between the enantiomers in their effects on force (P = 0.425) or [Ca2+](i) (P = 0.604). 4. The R(-)-enantiomer of ketamine is a more potent relaxant of ACh-induced ASM contraction than the S(+)-enantiomer. This difference appears to be caused by differential actions on receptor-operated calcium channels.