To compare the chondrogenic and proliferative effects of pooled human serum (HS) and fetal bovine serum (FBS) on tissue-engineered human nasal septal chondrocytes. Human chondrocytes were expanded for one passage in monolayer in medium supplemented with 10% FBS, 2% HS, 10% HS, or 20% HS. Cells were then suspended in alginate beads for 3D culture for 2 weeks with 10% FBS, 2% HS, 10% HS, or 20% HS. Monolayer cell yields were greater with HS than FBS. In alginate, cellular proliferation, glycosaminoglycan production per cell, and type II collagen were significantly higher with 10% HS compared to 10% FBS controls. HS results in increased proliferation and production of cartilaginous extracellular matrix by tissue-engineered human nasal septal chondrocytes, compared to FBS controls. Culture with human serum may facilitate creation of neocartilage constructs that more closely resemble native tissue.