Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Forster distance (R-0), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R-0 provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor-acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R-0 for BRET1 systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R-0 of the original BRET1 system. R-0 for BRET2 systems combining green fluorescent proteins (GFP(2)) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2-9% greater than the original BRET2 system despite being similar to 30-fold brighter. Crown Copyright (C) 2012 Published by Elsevier Inc. All rights reserved.