A series of N-terminal deletions of the mitochondrial targeting sequence of the HEM1 product, delta-aminolevulinate synthase, was constructed. Targeting ability of mutant signals was assayed in vivo by fusion to beta-galactosidase or to the mature delta-aminolevulinate synthase. In the former case, the subcellular location of the fusion proteins provided a measure of import efficiency. In the later case, constructs were tested for complementation of delta hem 1 strains. We found this complementation assay to be particularly sensitive if the delta hem 1 strain is rho-. The results of these experiments, together with experiments deleting the signal from the carboxyl end, indicate that the targeting information is encoded in non-overlapping regions of the HEM1 signal.