This study examined the effect of nicotine on fibroblast attachment to glass and nondiseased human root surfaces. Human foreskin fibroblasts (HFF) were trypsinized, suspended in RPMI 1640 medium, and incubated with autoclaved human root sections and nicotine concentrations of zero (control), 25, 50, 100, 200, or 400 ng/ml. The root sections were examined for fibroblast attachment at 24, 48, and 72 hours by light microscopy and scanning electron microscopy (SEM). Additional trypsinized HFF were incubated on glass surfaces with the same concentrations of nicotine and examined at one week by light microscopy. HFF attached and grew on glass and root surfaces at all concentrations of nicotine. Controls on glass surfaces exhibited a normal monolayer of long spindle-shaped fibroblasts with a parallel alignment and minimal overlapping. Nicotine-treated HFF exhibited a haphazard arrangement with cell overlapping and vacuolization of the cytoplasm. Under SEM, the controls had smooth surfaces and appeared firmly attached to the root surface via (1) microvilli and filopodia on the cell boundaries and (2) short, branched, thin-to-medium width cytoplasmic processes with microvilli and filopodia on their boundaries. Few microvilli were noted on the control cell surfaces. HFF exposed to nicotine had microvilli and filopodia on the cell surfaces and long thin and long broad cytoplasmic processes with many microvilli and filopodia that projected away from the root surface. These findings suggest that the nature of fibroblast attachment to glass and root surfaces is altered by nicotine. A similar disturbance in fibroblast attachment may occur in humans who use nicotine-containing products, making them more susceptible to destruction of the periodontium and less responsive to new attachment after periodontal therapy.