This study examined differences in accelerometer output when subjects walked on level ground and on a treadmill. We asked 25 nondisabled participants to wear an RT3 triaxial accelerometer (StayHealthy, Inc; Monrovia, California) and walk at their "normal" and "brisk" walking speeds for 10 minutes. These activities were repeated on a treadmill using the individual speeds from level-ground walking on two occasions 1 week apart. Paired t-tests found a difference in RT3 accelerometer vector magnitude (VM) counts/min between the two walking speeds on both surfaces on days 1 and 2 (p < 0.001). Although we found no significant differences between VM counts/min on the two surfaces at normal and brisk speeds on days 1 and 2 (p > 0.05), we found wide limits of agreement between level ground and treadmill walking at both speeds. Measurement and discrimination of walking intensity employing RT3 accelerometer VM counts/min on the treadmill demonstrated reasonable validity and stability over two time points compared with level-ground walking.