A new technique for generating extended pH gradients (3-4 pH units) in Immobiline gels for isoelectric separations is described. A five-chamber gradient mixer has been built, based on the 'Varigrad'-type mixers of Peterson and Sober (Anal. Chem. 31, 1959, 857-862). Each chamber contains one of the following Immobilines, in this order: pK values 4.4, 4.6, 6.2, 7.0 and 8.5, titrated in the pH 4-8 interval with non-buffering Immobilines pK 9.3 (in the case of the two acidic Immobilines) and pK 3.6 (in the case of the three basic Immobilines). In this way it is possible to cast, in a highly reproducible way, an immobilized pH gradient in the pH range 4.0 to 7.5, which should be ideal for isoelectric separations in the first dimension of two-dimensional techniques. A computer program is also described which, given the molarities and pK values of the different Immobilines in the chambers of the Varigrad mixer, can generate the theoretical pH profile, together with the buffering capacity (beta) and ionic strength (I) courses.