Two-dimensional polyacrylamide gel electrophoresis (PAGE), together with 2-D gel electrophoresis (GE) analysis software, is a common technique to analyze a complex proteome. In order to accurately locate the differentially expressed proteins in human pituitary macroadenoma tissues in our long-term research program to clarify the molecular mechanisms of macroadenoma formation, a reproducible separation system is needed. An immobilized pH-gradient dry gel-strip (IPG strip) has been extensively used for first-dimensional isoelectric focusing (IEF), and has achieved a high degree of reproducibility in the IEF direction. For the second dimension (SDS-PAGE), different types of gel systems are available, including horizontal vs. vertical gel systems, and gradient vs. constant-percentage gels. A typical horizontal system is the Multiphor II system that analyzes one gel at a time, using a precast gradient gel (180 x 245 x 0.5 mm), and a typical vertical system is the Dodeca system, which analyzes up to 12 gels at a time, using usually a single-concentration gel (190 x 205 x 1 mm). The present study evaluated the spatial and quantitative reproducibility of the two systems for the separation of the complex human pituitary proteome. PDQuest software was used to analyze the digitized gel-image data, and SPSS statistical software was used to analyze the data. The results demonstrated a high percentage (>99%) of protein-spot matches within each electrophoretic system. The Dodeca gel system demonstrated better between-gel reproducibility for spot position, higher resolution in the Sodium dodecyl sulfate (SDS)-PAGE direction, lower gel background, better spot quality, and higher reproducibility of the spot volume.