We have engineered a mutant version of the green fluorescent protein GFP (Cormack et al. Selected for bright fluorescence in E. coli. Gene 1996;173:33-38) for expression in the protozoan parasite Toxoplasma gondii. Although intact GFP was not expressed at any detectable level, GFP fusion proteins could be detected by fluorescence microscopy, flow cytometry (FACS), and immunoblotting. Both extracellular tachyzoites and T. gondii-infected host cells could readily be sorted by FACS, which should facilitate a variety of selection strategies. Several selectable markers were tested for their ability to produce stable green transgenic parasites. Fluorescence intensity was directly correlated with gene copy number and protein expression level. Weak selectable markers such as chloramphenicol acetyl transferase (CAT) driven by the SAG1 promoter, which yield multicopy insertions, are therefore most effective for selecting green fluorescent parasites-particularly when coupled to constructs which employ a strong promoter to drive GFP expression. Transformation vectors developed in the course of this work should be of general utility for the overexpression of heterologous transgenes in Toxoplasma. CAT-GFP fusion proteins were expressed in the parasite cytoplasm. GFP fusions to the P30 major surface antigen (linked on the same plasmid to a CAT selectable marker under control of various promoters) could be detected in dense granules within living cells, and were efficiently secreted into the parasitophorous vacuole. GFP fusions to the rhoptry protein ROP1 were targeted to rhoptries (specialized secretory organelles at the apical end of the parasite).