Isoelectric focusing (IEF) of human serum transferrin allows splitting of the protein pattern into three forms corresponding to the diferric, monoferric and apoform. A detailed analysis of this pattern, performed on transferrin at different degrees of iron saturation, demonstrated that free Ampholine carrier ampholytes (CA) alter the expected results, always giving a complex pattern with multiple bands. In particular, the monoferric form appears to be the predominant one, regardless of the starting saturation of transferrin. In contrast to IEF-CA, the new technique of IEF in immobilized pH gradients (IPG), shows a much simpler pattern with the same samples. Moreover, the different transferrin forms are focused at the same pI values as in IEF-CA but the pattern appears to correspond to the expected distribution. IPG analysis gives a pattern similar to IEF-CA when free Ampholine CA are added either to the samples and/or as electrode solutions. A chelating action of Ampholine CA on Fe+3 might be responsible for these effects, while Immobilines, due to their different chemical nature or to the different focusing procedure, are not able to interact with iron.