Unusual C 35 to C 38 alkenones were identified in mid-Holocene (8-3.5 kyr BP) sediments from a restricted estuary in southwest Florida (Charlotte Harbor). The distribution was dominated by a C 36 diunsaturated (ω15,20) ethyl ketone, identical to the one present in Black Sea Unit 2 sediments. Other unusual alkenones were tentatively assigned as a C 35:2 (ω 15,20) methyl ketone, a C 37:2 (ω 17,22) methyl ketone and a C 38:2 (ω 17,22) ethyl ketone. In late Holocene sediments < 3.5 kyr BP, the common C 37 to C 39 alkenones were found. Compound-specific 14 C, 13 C, and D isotope measurements were used to constrain the possible origin of the alkenones. Conventional radiocarbon ages of alkenones and higher plant-derived long chain n-alcohols indicated no significant difference in age between mid-Holocene alkenones and higher plant n-alcohols. Both alcohols and alkenones were offset vs. calibrated ages of shell fragments in the same sediment core, which suggests they were pre-aged by 500-800 yr, implying resuspension and redistribution of the fine-grained sedimentary particles with which they are associated. The hydrogen isotopic (δD) composition (-190‰ to -200‰) of the C 37 and C 38 alkenones in the late Holocene sediments is in line with values for coastal haptophytes in brackish water. However, the unusual C 36 and C 38 alkenones from the mid Holocene sediments were enriched in D (by ca. 100‰) vs. the late Holocene alkenones. Also, δ 13 C values of mid-Holocene alkenones were consistently offset compared with late Holocene alkenones (-21‰ to -22‰ and -22‰ to -23‰, respectively). We suggest that the alkenones in Charlotte Harbor were produced by unknown alkenone-producing haptophyte.