Diatoms are unicellular photoautotrophic eukaryotes that play an important role in ecology by fixing large amounts of CO₂ in the oceans. Because they evolved by secondary endocytobiosis—a process of uptake of a eukaryotic alga into another eukaryotic cell—they have a rather unusual cell biology and genetic constitution. Because the preparation of organelles is rather difficult as a result of the cytosolic structures, genetic transformation and expression of preproteins fused to green fluorescent protein (GFP) became one of the major tools to analyze subcellular localization of proteins in diatoms. Meanwhile several groups successfully attempted to develop genetic transformation protocols for diatoms. These methods are based on ‘‘biolistic’’ DNA delivery via a particle gun and allow the introduction and expression of foreign genes in the algae. Here a protocol for the genetic transformation of the diatom Phaeodactylum tricornutum is described as well as the subsequent characterization of the transformants.