Lecithin:cholesterol acyltransferase (LCAT) is crucial to the maturation of high‐density lipoprotein (HDL). Homozygosity for LCAT mutations underlies rare disorders characterized by HDL‐cholesterol (HDL‐c) deficiency while heterozygotes have half normal HDL‐c levels. We studied the prevalence of LCAT mutations in referred patients with low HDL‐c to better understand the molecular basis of low HDL‐c in our patients. LCAT was sequenced in 98 patients referred for HDL‐c <5th percentile and in four patients referred for low HDL‐c and corneal opacities. LCAT mutations were highly prevalent: in 28 of the 98 participants (29%), heterozygosity for nonsynonymous mutations was identified while 18 patients carried the same mutation (p.T147I). The four patients with corneal opacity were compound heterozygotes. All previously identified mutations are documented to cause loss of catalytic activity. Nine novel mutations—c.402G>T (p.E134D), c.403T>A (p.Y135N), c.964C>T (p.R322C), c.296G>C (p.W99S), c.736G>T (p.V246F), c.802C>T (p.R268C), c.945G>A (p.W315X), c.1012C>T (p.L338F), and c.1039C>T (p.R347C)–‐were shown to be functional through in vitro characterization. The effect of several mutations on the core protein structure was studied by a three‐dimensional (3D) model. Unlike previous reports, functional mutations in LCAT were found in 29% of patients with low HDL‐c, thus constituting a common cause of low HDL‐c in referred patients in The Netherlands. Hum Mutat 32:1290–1298, 2011. ©2011 Wiley Periodicals, Inc.