We assessed age-related differences in adults in familiarization to treadmill walking within virtual environments (VE), and examined whether treadmill walking after familiarization resembles overground walking. Seventeen younger and 17 older adults walked at preferred speed on an overground walkway and afterwards walked at the same speed for 20 min on a treadmill coupled to a VE. A motion capture system was used to measure spatio-temporal gait parameters. On the treadmill, both younger and older adults initially displayed decreased step length and increased step width, cadence, and time in double support relative to overground walking. Except for time in double support, step characteristics approached overground walking-behavior with a negatively accelerated trend. After 15 min of treadmill walking, changes became minor corresponding to less than 1% deviations to individuals' overground walking. At the end of familiarization, average differences in step length, cadence, and double support relative to overground walking were reduced to less than 5 percent in both age groups. For step width, younger adults approximated overground walking after 20 min more closely than older adults, probably reflecting larger initial differences between treadmill and overground walking among older adults. We conclude (a) that 20 min of familiarization to treadmill walking in a VE are sufficient to reach stable walking patterns resembling those observed in overground walking, but that some differences between the two settings remain, especially in older adults; (b) that sufficient familiarization to the treadmill is needed to ascertain the validity and generalizability of comparisons between younger and older adults. (C) 2009 Elsevier B.V. All rights reserved.