Fungal cells are highly complex as their metabolism is compartmentalized harboring various types of subcellular organelles that are bordered by one or more membranes. Knowledge about the intracellular localization of transporter proteins is often required for the understanding of their biological function. Among different approaches available, the localization analysis based on the expression of GFP fusions is commonly used as a relatively fast and cost-efficient method that allows visualization of proteins of interest in both live and fixed cells. In addition, inactivation of transporter genes is an important tool to resolve their specific function. Here we provide a detailed protocol for the deletion and localization analysis of ABC transporters in the filamentous fungus Penicillium chrysogenum. It includes construction of expression plasmids, their transformation into fungal strains, cultivation of transformants, microscopy analysis, as well as additional protocols on staining of fungal cells with organelle-specific dyes like Hoechst 33342, MitoTracker DeepRed, and FM4-64.