We have examined the biological properties of CEA6, a human carcinoembryonic antigen (CEA)-specific single-chain Fv (scFv) isolated by phage display, and five related clones derived by affinity maturation and selected for improved off-rate (Koff). All clones bind strongly and specifically to CEA-positive human tumours by immunocytochemistry and show negligible cross-reactivity with normal colon. Flow cytometry of scFv on human liver cells indicates a shift in fine epitope specificity resulting from mutagenesis. All monomeric scFv have been radioiodinated, retaining effectively full binding activity. A single intravenous injection into nude mice bearing human colon tumour xenografts confirms tumour targeting in all cases. As reported in other studies, the kidney is the main route of elimination of scFv at early time points. Tumour binding of the parental antibody CEA6 consistently gives the highest tumour-blood ratios at 24 h (mean 16:1). Clone TO6D11, which has a sevenfold reduced Koff relative to CEA6, showed no difference in tumour uptake at 24 h but persisted at the tumour site for longer than CEA6. This study demonstrates a possible correlation between binding affinity and tumour residence time when examined in this model.