The peroxisome biogenesis disorders (PBD) are a heterogeneous group of lethal, autosomal recessive diseases that are characterized by defective import of peroxisomal matrix proteins. Zellweger syndrome (ZS) is the most severe phenotype, neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD) are milder. Based on homology to yeast peroxisome assembly genes, we identified two genes that cause PBD. Here we report analysis of the patient mutations, explain the phenotypes and provide novel information on protein function. Defects in the peroxisome targeting signal (PTS) 1 receptor gene, PXR1, define complementation group (cg) 2 (Nature Genetics 9:115-124, 1995). In contrast to the yeast PTS1 receptor mutants, which are defective only in PTS1 import, one of the two patients (PBD 005) was defective in the import of both PTS1 and PTS2 proteins into peroxisomes. We show that there are two classes of PXR1 transcripts that differ by an alternatively spliced exon: PXR1L contains the exon, PXR1S does not. Fibroblasts from PBD 005 (ZS, homozygous R390ter) have greatly reduced levels of PXR1 mRNA and no detectable Pxr1p, whereas fibroblasts from PBD 018 (NALD, homozygous N489K), show normal levels ofPXR1 mRNA and protein. Transfection of PBD 005 fibroblasts with either PXR1L or PXR1S cDNA restores PTS1 mediated import, but onlyPXR1L corrects the PTS2 import defect. These results indicate that in mammals, in contrast to yeast, there is direct interaction between the two peroxisomal import pathways. Additionally, we have identified a second gene,PXAAA1, predicted to encode a member of the AAA family of putative ATPases. We show that mutations in PXAAA1 account for cg4 of the PBD(the second largest cg, with all three classical phenotypes) and that the encoded protein is required for the stability of PXR1p.