The desilked silkworm (Bombyx mori L.) pupae oil is a good source of a-linolenic acid (ALA), but selectively enriching ALA is a great technical challenge due to the presence of multiple analogues such as linoleic acid and oleic acid. To set up a simple, highly selective and low-cost combinative inclusion process for obtaining ALA concentrate from silkworm pupae oil, the effects of b-cyclodextrin (b-CD) inclusion parameters on the purity and recovery of polyunsaturated fatty acid (PUFA) were investigated; then, four types of combinative processes using b-CD and/or urea were compared. The results indicated that a two-stage combinative inclusion process was established. In the first stage, when the volume ratio of b-CD-saturated solution to free fatty acids ethanol solution was 9 v/v, the dispersion temperature and time were 608C and 1.5 h, the freezing temperature and time were À108C and 15 h, and the purity of ALA was raised from 15.0 to 47.6% with a recovery of 39.4%. Then, the purity of ALA was further increased to 67.4% in the secondstage operation with a corresponding recovery of 34.8% using urea complexation. Abbreviations: b-CD, b-cyclodextrin; ALA, a-linolenic acid; CICC, combinative inclusion of b-CD and b-CD; CICU, combinative inclusion of b-CD and urea; CIUC, combinative inclusion of urea and b-CD; CIUU, combinative inclusion of urea and urea; EPA, eicosapentaenoic acid; FFA, free fatty acids; LA, linoleic acid; LCPUFA, long chain PUFA; OA, oleic acid; PA, palmitic acid; PUFA, polyunsaturated fatty acid; SA, stearic acid Eur.