Protein arrays make possible the functional screening of large numbers of immobilized proteins in parallel. To facilitate the supply of proteins and to avoid their deterioration on storage, we describe our protein in situ array (PISA) method for production of protein arrays in a single step directly from PCR DNA, using cell-free transcription and translation. In PISA, the in vitro-generated proteins are immobilized, as they are formed, on the surface of wells, beads, or slides coated with a protein-capturing reagent. In our preferred method, proteins are tagged with a double-hexahistidine sequence that binds strongly to Ni-NTA-coated surfaces. Advantages of PISA include avoiding bacterial expression and protein purification and making functional protein arrays available as required from genetic information.