A new nonlinear immobilized pH gradient (IPG) is proposed as the first dimension for two-dimensional electrophoresis. In comparison to conventional carrier ampholyte techniques, it offers better resolution and greater reproducibility whilst allowing application of higher protein loads. Furthermore, we have checked and supplemented existing data on pK values for the immobilized groups in the presence of 8M urea. This is necessary for pH gradients to be defined in a pH scale relevant to the focusing conditions such that spot positions can be related to amino acid compositions. The data will allow definition of pH scales for the temperature range 10-25 degrees C and for a pH range covering the major part of the nonlinear pH gradient. With the latter, focusing positions are neither influenced by urea concentration nor by the choice or the concentration of detergent or carrier ampholyte. Temperature is the only parameter affecting focusing reproducibility and here any changes in focusing positions can be related to the amino acid compositions of peptides.