A two-dimensional (2-D) electrophoresis procedure was optimized to obtain well-resolved and reproducible patterns of zein polypeptides in maize. All zein isoforms obtained through zein-specific extraction were observed from whole meal extracted with a urea/Triton/2-mercaptoethanol solution. Loading the protein samples at the acidic side of the gradient, using an amino acid solution as catholyte and running for a short time period under high Vh reduced shrinking and instability at the basic side of the isoelectric focusing (IEF) gels. Good quality and reproducible 2-D patterns were thus obtained, allowing automatic spot quantification. A linear relationship between spot intensity and zein isoform amount was established for 20 of 22 zeins detected in a 5.5-8.5 pH range using colloidal Coomassie blue staining in one maize line. The analysis of 45 genetically diverse inbred lines allowed the detection of 59 isoforms belonging to the four classes of zeins, and revealed a large qualitative and quantitative variability of individual isoforms. The classical decrease in zein amount in o2 mutant genotype was observed, and could be quantified for every isoform. The improved technique will be useful to dissect the genetic control of zein expression in maize.