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The theoretical predictions of the triple isotopic substitution (TIS) method in small-angle neutron scattering have been confirmed experimentally. A unique advantage of the method, namely the possibility to make part of a complex particle 'invisible' in normal (H20) buffer, is demonstrated. Particle association, the addition of 'small' molecules (such as glycerol) or 'large' molecules (such as pyruvate kinase) in the solutions under study have been shown not to influence the difference scattering curve obtained. The possibility of extracting normalized interparticle interference functions from the TIS data is discussed. This enables the detection of the presence of aggregates (e.g. dimers) in the solution, even in cases where the molar mass of a particle and/or its concentration are unknown. A simple internal test is suggested which checks whether or not sufficient conditions for the applicability of the method are fulfilled.

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