The aim of this study was to investigate the effect of rearing substrate and space allowance in the home environment on the motivation of dairy calves to perform locomotor play in an arena test. At 1wk of age, 72 calves were moved into one of 18 experimental pens (n=4 calves/pen) where they remained until 6wk of age. Half of the pens had floors covered with stones (ST) and the other half were covered with sawdust (SD). For each substrate type, calves were reared at one of three space allowances: 1.0, 1.5 or 2.0m2/calf. Behaviour was video recorded continuously for 24h in the home environment at 4 and 6wk of age. Calves were tested individually in an arena test at 3 and 6wk of age, where behaviour was recorded for 20min. The arena floor was covered in wood chip. Running duration and the frequency of running, jumping, kicking and bucking were scored continuously and used as measures of locomotor play. At 4 and 6wk of age in the home environment, calves reared on ST spent less (P<0.05) time walking than calves reared on SD and calves reared at 1.0m2/calf spent less time (P<0.05) walking than calves reared at 1.5 or 2.0m2/calf. However, substrate type and space allowance did not affect (P>0.05) time calves spent running in the home environment. In the test arena, calves reared on ST ran more at 3 (1.7 and 1.5±0.05 square-root transformed values, ST and SD respectively, P=0.001) and 6 (1.4 and 1.2±0.08 square-root transformed values, ST and SD respectively, P=0.035) wk of age than calves reared on SD. At 3wk of age, calves reared at 1.0m2/calf spent more (P=0.016) time running than calves reared at 2.0 m2/calf (1.7, 1.6 and 1.5±0.06 square-root transformed values, 1.0, 1.5 and 2.0m2/calf respectively, P=0.022) in the test arena. Furthermore, time spent running in the home pen was positively correlated with running duration in the arena test irrespective of substrate type (ST: r=0.35, P=0.003 and SD: r=0.32, P=0.008) and space allowance (1.0m2/calf: r=0.42, P=0.004; 1.5m2/calf: r=0.28, P=0.054; 2.0m2/calf: r=0.25, P=0.095). Overall, our results suggest that different housing conditions (rearing substrate and space allowance) can affect the motivation of calves to perform locomotor play in an arena test, however the stimuli associated with this increased motivation to play remains unclear.