The fluorescent proteins ECFP and HcRed were shown to give an easily resolved FRET-signal when expressed as a fusion inside mammalian cells. HeLa-tat cells expressing ECFP, pHcRed, or the fusion protein pHcRed-ECFP were analyzed by flow cytometry after excitation of ECFP. Cells expressing HcRed-ECFP, or ECFP and HcRed, were mixed and FACS-sorted for FRET positive cells: HcRed-ECFP cells were greatly enriched (72 times). Next, cloned human antibodies were fused with ECFP and expressed anchored to the ER membrane. Their cognate antigens (HIV-1 gp120 or gp41) were fused to HcRed and co-expressed in the ER. An increase of 13.5 +/- 1.5% (mean +/- SEM) and 8.0 +/- 10.7% in ECFP fluorescence for the specific antibodies reacting with gp 120 or gp41, respectively, was noted after photobleaching. A positive control (HcRed-ECFP) gave a 14.8 +/- 2.6% increase. Surprisingly, the unspecific antibody (anti-TT) showed 12.1 +/- 1.1% increase, possibly because overexpression in the limited ER compartment gave false FRET signals. (c) 2006 Elsevier Inc. All rights reserved.