Systemic primary carnitine deficiency (CDSP) is caused by recessive mutations in the SLC22A5 (OCTN2) gene encoding the plasmalemmal carnitine transporter and characterized by hypoketotic hypoglycemia, and skeletal and cardiac myopathy. The entire coding regions of the OCTN2 gene were sequenced in 143 unrelated subjects suspected of having CDSP. In 70 unrelated infants evaluated because of abnormal newborn screening (NBS) results, 48 were found to have at least 1 mutation/unclassified missense variant. Twenty-eight of 33 mothers whose infants had abnormal NBS results were found to carry at least 1 mutation/unclassified missense variant, including 11 asymptomatic mothers who had 2 mutations. Therefore, sequencing of the OCTN2 gene is recommended for infants with abnormal NBS results and for their mothers. Conversely, 52 unrelated subjects were tested due to clinical indications other than abnormal NBS and only 14 of them were found to have at least one mutation/unclassified variant. Custom designed oligonucleotide array CGH analysis revealed a heterozygous ∼1.6 Mb deletion encompassing the entire OCTN2 gene in one subject who was apparently homozygous for the c.680G>A (p.R227H) mutation. Thus, copy number abnormalities at the OCTN2 locus should be considered if by sequencing, an apparently homozygous mutation or only one mutant allele is identified. ©2010 Wiley-Liss, Inc.