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Most cells synthesize their glycerophospholipids and triglycerides (TG) to maintain the cellular integrity and to provide energy for cellular functions. The phospholipids are synthesized de novo in cells through an evolutionary conserved process involving serial acylations of glycerol-3-phosphate. Several isoforms of the enzyme 1-acylglycerol-3-phosphate-O-acyltransferase (AGPAT) acylate lysophosphatidic acid at the sn-2 position to produce phosphatidic acid. We cloned a cDNA predicted to be an AGPAT isoform and designated it AGPAT9. The human AGPAT9 gene spans across 14 exons and encodes for a polypeptide of 534 amino acids. AGPAT9 is highly expressed in the lung and spleen, followed by leukocyte, omental adipose tissue, and placenta. In the Chinese Hamster Ovary (CHO), cell lysates overexpressing AGPAT9, we observed AGPAT activity but not the lysophosphatidylcholine acyltransferase activity. When AGPAT9 is coexpressed with AGPAT1 in CHO cells, both the isoforms localize to the endoplasmic reticulum (ER) and occupy the same ER domain as AGPAT1. Despite substitution of asparagine with proline in the NHX(4)D motif and arginine with cysteine in the EGTR motif, AGPAT9 retains AGPAT activity suggesting that residues asparagine and arginine in the NHX(4)D and EGTR motifs respectively are not essential for the enzymatic activity. Based on the X-ray crystallographic structure of a related acyltransferase, squash gpat, a model is proposed in which a hydrophobic pocket in AGPAT9 accommodates fatty acyl chains of both substrates in an orientation, whereas the HX(4)D motif participates in catalysis. Based on the activity and expression pattern of AGPAT9 in the lung and spleen, this novel isoform could be implicated in the biosynthesis of phospholipids and TG in these tissues.