The human dental follicle partially differentiates into the periodontal ligament (PDL), but their biological functions are different. The gene-expression profiles of the dental follicle and PDL were compared using the cDNA microarray technique. Microarray analysis identified 490 genes with a twofold or greater difference in expression, 365 and 125 of which were more abundant in the dental follicle and PDL, respectively. The most strongly expressed genes in the dental follicle were those related to bone development and remodeling (EGFL6, MMP8, FRZB, and NELL1), apoptosis and chemotaxis (Nox4, CXCL13, and CCL2), and tooth and embryo development (WNT2, PAX3, FGF7, AMBN, AMTN, and SLC4A4), while in the PDL it was the tumor-suppressor gene WIF1. Genes related to bone development and remodeling (STMN2, IBSP, BMP8A, BGLAP, ACP5, OPN, BMP3, and TM7SF4) and wound healing (IL1, IL8, MMP3, and MMP9) were also more strongly expressed in the PDL than in the dental follicle. In selected genes, a comparison among cDNA microarray, real-time reverse-transcription polymerase chain reaction, and immunohistochemical staining confirmed similar relative gene expressions. The gene-expression profiles presented here identify candidate genes that may enable differentiation between the dental follicle and PDL.