The phospholipid fatty acid composition of the NorthEast Atlantic sponge Polymastia penicillus (South Brittany, France) was investigated. Sixty fatty acids (FA) were identified as methyl esters (FAME) and N-acyl pyrrolidides (NAP) by gas chromatography-mass spectrometry (GC/MS), including eight D5,9 unsaturated FA and three long-chain 2-hydroxylated FA. The major phospholipid FA were palmitic (14.3% of the total FA mixture), vaccenic (12.7%), 15(Z)-docosenoic (13.4%) and 5(Z),9(Z)-hexacosadienoic (13.3%) acids. In addition to the iso-and anteiso-branched saturated FA, several unusual short-chain branched saturated FA were identified. In addition to the known D5,9 FA, and interestingly regarding their identification by GC-MS as N-acyl pyrrolidides, was the co-occurrence of unusual FA possessing a D3, D4 and D5 double bond such as iso-4-pentadecenoic, iso-5-heptadecenoic, anteiso-5-heptadecenoic and two new compounds, not hitherto found in nature, namely 17methyl-13-octadecenoic (0.8%) and 3,16-docosadienoic (1.1%) acids.