Abstract Forest humus layers derived from Norway spruce [ Picea abies (L.) Karst.] were fractionated into unbound lipid (UL), unbound polar compounds (UPC), hydrolyzable compounds (HY), bound lipid (BL). bound polar compounds (BPC) and residue (RES) by a combination of solvent extraction and acid hydrolysis. On a dry weight basis the UL and BL fractions totalled 7.7 and 3.0%, respectively, of the spruce litter material (L layer), but decreased markedly to 0.7 and 0.3% in the mineral soil (Aeh horizon). Unbound and bound lipid C together amounted to 14.8% of total organic C in the L and 13.6% in the Aeh horizon, showing a minimum in the Of (11.6%) and Oh (11.4%) layers. This chemically-detectable alkyl-C (sum of UL- and BL-C) accounted for two-thirds of the spruce litter total alkyl-C estimated from solid state (CPMAS) 13C-NMR spectra, but only for one-third of the mineral soil alkyl-C. Spruce litter UL contained higher amounts of C, O and H compared to BL, and with increasing depth significant changes in C, O and H contents were observed for both lipid fractions. C-to-H atomic ratios were consistently lower for UL (7.3–7.7) than BL (8.2–9.0), whereas unbound and bound lipid C-to-O ratios were nearly identical in spruce litter (4.7–4.8), but differed greatly in the Aeh horizon (UL: 5.5; BL: 3.1). Preparative column chromatography on silica gel of unbound lipid separated a series of subfractions, preliminary labelled as less polar lipid. glycolipid, phospholipid and highly polar lipid. Glycoplipid was the dominant component of spruce litter unbound lipid (45%) and further increased in the Aeh horizon (60%), while less polar lipid showed highest percentages in the L (41%) and Oh layers (42%). Phospholipid (9–15%) and highly polar lipid (2–7%) were generally minor lipid subfractions in the forest humus profile.