Rhizomelic chondrodysplasia punctata (RCDP) is a disorder of peroxisome metabolism result-ing from a deficiency of plasmalogens, a specialized class of membrane phospholipids. Classically, patients have a skeletal dysplasia and profound mental retardation, al-though milder phenotypes are increasingly being iden-tified. It is commonly caused by defects in the peroxi-some transporter, PEX7 (RCDP1), and less frequently due to defects in the peroxisomal enzymes required to initi-ate plasmalogen synthesis, GNPAT (RCDP2) and AGPS (RCDP3). PEX7 transports AGPS into the peroxisome, where AGPS and GNPAT partner on the luminal mem-brane surface. The presence of AGPS is thought to be required for GNPAT activity. We present six additional probands with RCDP2 and RCDP3, and the novel muta-tions identified in them. Using cell lines from these and previously reported patients, we compared the amounts of both AGPS and GNPAT proteins present for the first time. We used protein modeling to predict the structural consequences ofAGPSmutations and transcript analysis to predict consequences ofGNPATmutations, and show that milder RCDP phenotypes are likely to be associated with residual protein function. In addition, we propose that full GNPAT activity depends not only on the pres-ence of AGPS, but also on the integrity of substrate chan-neling from GNPAT to AGPS.