We have examined the effect of oleic acid (OA) concentrations and incubation time, along with chicken serum (CS), on adipogenic differentiation and expression of adipogenic transcripts in hen preadipocytes. Preadipocytes were treated with (i) an adipogenic cocktail (DMI) containing 500nM dexamethasone, 0.5mM 3-isobutyl-1-methylxanthine and 20 mu g/mL insulin alone and DMI+75, 150, 300 or 600 mu M OA for 48h; (ii) DMI+300 mu M OA (DMIOA) for 6, 12, 24 or 48h; and (iii) foetal bovine serum (FBS), CS, DMI+FBS, DMI+CS, DMIOA+FBS and DMIOA+CS. While FABP4 was significantly expressed with increasing concentrations of OA, the expression of C/EBP, LEPR and FAS were unchanged compared with the control. PPAR2 expression was unchanged across all time-points. A significantly higher level of C/EBP was measured at 48h, but the levels of C/EBP increased after 12h. Levels of FABP4 significantly increased with the time of incubation after 12h, but that of LPL was reduced (P<0.05) at 6, 24 and 48h. FABP4 was highly expressed in cells treated with CS, DMI+CS and DMIOA+CS compared to cells treated with FBS, DMI+FBS and DMIOA+FBS. In conclusion, increased concentrations of OA and incubation time increases lipid accumulation; FABP4 and C/EBP are potential transcription factors regulating OA induced adipogenesis of fat cells obtained from laying hen. CS is a potent inducer of adipogenic differentiation in hen preadipocytes.