The protozoan parasite Toxoplasma gondii is a highly specialized eukaryote that contains a remarkable number of intracellular compartments, some unique to Apicomplexans and others typical of eukaryotes in general. We have established a green fluorescent protein (GFP)-based motif-trap to identify proteins targeted to different intracellular locations and subsequently the signals responsible for this sorting. The motif-trap involves the transfection and integration of a linearized GFP construct which lacks a promoter and an initiator methionine codon. FACS is used to isolate parasites in which GFP fuses in-frame into a coding region followed by screening by fluorescence microscopy for those containing GFP targeted to specific intracellular compartments. GFP trapping was successful using vectors designed for integration into regions encoding exons and vectors that were engineered with a splice acceptor site for integration into regions encoding introns. This strategy differs from most protein traps in that the resulting fusions are expressed from the endogenous promoter and starting methionine. Thus, problems from inappropriate expression levels or the creation of fortuitous targeting signals seen in library-based traps are diminished. Using this approach, we have trapped GFP localized to a number of intracellular compartments including the nucleus, nucleolus, endoplasmic reticulum, cytosol, parasite surface and rhoptries of Toxoplasma. Further analysis of a parasite clone containing GFP targeted to the rhoptries shows GFP fused to the gene encoding the rhoptry protein ROP4 and has elucidated an additional mechanism for targeting of this protein.