Reflection high-energy electron diffraction and scanning tunnelling microscopy (STM) have been used to study InAs/GaAs quantum dot (QD) formation in bilayer QD structures grown by molecular beam epitaxy on GaAs(0 0 1) substrates. For GaAs spacer layers thicknesses < 200 Angstrom, there is a reduction in the critical coverage (theta(crit)) required to form the second layer QDs, the change in theta(crit) compared with first layer growth depending on the size of the QDs in the initial layer. STM measurements of the uncapped QDs show that second layer dots grown on thick GaAs spacer layers (> 200 Angstrom) are identical to those grown in the first layer. By contrast, QD formation on a thin spacer layer (< 200 Angstrom) results in a lower density of larger QDs that nucleate preferentially on top of the mounds that dominate the morphology of the relatively thin GaAs capping layer. The introduction of an annealing step before second layer QD growth smooths the spacer layer surface morphology but has no effect on theta(crit). The implication is that strain is the dominant cause of the observed variation in second layer theta(crit). (C) 2002 Elsevier Science B.V. All rights reserved.