Plant non-specific lipid-transfer proteins (nsLTPs) have received an increasing interest as potential drug carriers in drug delivery systems. NsLTPs are subdivided into nsLTP1 (9 kDa) and nsLTP2 (7 kDa) according to the molecular weight. All of nsLTPs are highly stable proteins because they possess eight highly conserved cysteine residues forming four disuffide bonds. These highly stable proteins can protect drugs against oxidation or degradation. In this paper, the application of nsLTPs in a drug carrier system was comprehended through scanning chemical compounds to obtain the potential nsLTPs-binding drugs from the comprehensive medicinal chemistry (CMC) database. These results helped us to realize the binding differences for preferred drugs between maize nsLTP1 and rice nsLTP2. We have successfully constructed a rice nsLTP2 mutant (Y45W) to improve fluorescence sensitivity. The fluorescence binding assay showed that nsLTP2 can associate with sterol-like or triphenylmethane-like molecules but the binding affinities of nsLTP2 with both of nsLTP2-binding drug candidates are quite different. Dissociation constants (Kd) for sterol/nsLTP2 complexes is below one micromolar and it is sufficient for these molecules to slowly release in a controlled-release drug delivery process. In addition, titration curve shows that binding model for nsLTP2 with the triphenylmethyl moiety of the molecule is more complicated. The basic triphenyl ring system may be critical for the nsLTP2 association. These results imply that rice nsLTP2 have highly potential applications in pharmaceuticals. The procedure combined a unique computer-based high throughput screening (HTS) method with an experimental binding assay, can effectively determine potential nsLTPs-binding drugs from the compound library, thus increasing the added value of nsLTPs in a drug carrier system. (C) 2004 Elsevier Inc. All rights reserved.