Long-chain fatty acids (FAs) stimulate clonal preadipocyte differentiation with α-linolenic acid (C18:3) being very potent. Porcine preadipocytes were differentiated for 24 h with 0, 50, 100, or 300 μM oleic acid (C18:1), linoleic acid (C18:2), or C18:3. Differentiation was increased to a greater extent by C18:1 and C18:2 than by C18:3 (P<0.01) suggesting species differences. The increase in mRNA for the transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT-enhancer binding protein alpha, and for the adipocyte-characteristic protein, lipoprotein lipase was less than twofold in all cases. Increased differentiation with small increases in PPARγ mRNA suggests the FAs provided ligand to activate PPARγ already present at initiation of differentiation when ligand synthesis was minimal. Fatty acid binding protein mRNA was increased several-fold by all three FAs. If these results extrapolate to the intact animal, they suggest the dietary FA composition will differentially affect fat deposition in the pig. Abbreviations: aP2, adipocyte fatty acid binding protein; C/EBPα, CCAAT-enhancer binding protein alpha, C/EBPβ, CCAAT-enhancer binding protein beta; C/EBPδ, CCAAT-enhancer binding protein delta; C18:0, stearic acid; C18:1, oleic acid, C18:2, linoleic acid, C18:3, α-linolenic acid; C20:4, arachidonic acid; C22:6, docosaheaxaenoic acid; 9,11-CLA, cis 9, trans 11-conjugated linoleic acid; DMEM, Dulbecco’s modified Eagle’s medium; 18S, 18S ribosomal RNA; FA, long-chain fatty acid; F12, nutrient mixture F-12 Ham; LPL, lipoprotein lipase; PPARγ, peroxisome proliferator-activated receptor gamma; PUFA, polyunsaturated fatty acid; RXRα, retinoid x receptor alpha.