As the Cne PRP8 intein is active and exists in an essential gene of an important fungal pathogen, inhibitors of splicing and assays for intein activity are of interest. The self-splicing activity of Cne PRP8, the intein from the Prp8 gene of Cryptococcus neoformans, was assessed in different heterologous fusion proteins expressed in Escherichia coli. Placement of a putatively inactive variant of the intein adjacent to the α-complementation peptide abolished the peptide's ability to restore β-galactosidase activity, while an active variant allowed complementation. This α-complementation peptide therefore provides a facile assay of splicing which can be used to test potential inhibitors. When placed between two heterologous protein domains, splicing was impaired by a β-branched amino acid immediately preceding the intein, while splicing occurred only with a hydroxyl or thiol immediately following the intein. Both these assays sensitively report impairment of splicing and provide information on how context constrains the splicing ability of Cne PRP8.