The introduction of novel proteins into foods carries a risk of eliciting allergic reactions in individuals sensitive to the introduced protein. Therefore, decision trees for evaluation of the risk have been developed, the latest being proposed by WHO/FAO early in 2001. Proteins developed using modern biotechnology and derived from fish are being considered for use in food and other applications, and since allergy to fish is well established, a potential risk from such proteins to susceptible human beings exists. The overall aim of the study was to investigate the potential allergenicity of an Ice Structuring Protein (ISP) originating from an arctic fish (the ocean pout, Macrozoarces americanus) using the newly developed decision tree proposed by FAO/WHO. The methods used were those proposed by FAO/WHO including amino acid sequence analysis for sequence similarity to known allergens, methods for assessing degradability under standardised conditions, assays for detection of specific IgE against the protein (Maxisorb RAST) and histamine release from human basophils. In the present paper we describe the serum screening phase of the study and discuss the overall application of the decision tree to the assessment of the potential allergenicity of ISP Type III. In an accompanying paper [Food Chem. Toxicol. 40 (2002) 965], we detail the specific methodology used for the sequence analysis and assessment of resistance to pepsin-catalysed proteolysis of this protein. The ISP showed no sequence similarity to known allergens nor was it stable to proteolytic degradation using standardised methods. Using sera from 20 patients with a well-documented clinical history of fish allergy, positive in skin prick tests to ocean pout, eel pout and eel were used, positive IgE-binding in vitro to extracts of the same fish was confirmed. The sera also elicited histamine release in vitro in the presence of the same extracts. The ISP was negative in all cases in the same experiments. Using the proposed decision tree, we demonstrated the safety of the ISP to patients already sensitised to fish, as well as to individuals potentially susceptible to producing IgE responses to proteins. Furthermore, the practicability of the new decision tree was confirmed.