Free amino acid concentrations and nitrogen (N) isotopic composition in new current-year (new), mature current-year (middle-aged) and previous-year (old) Masson pine (Pinus massoniana Lamb.) needles were determined to indicate atmospheric N deposition in Guiyang (SW China). In different areas, free amino acids (especially arginine) concentrations in new and middle-aged needles were higher than in old needles, and the variation of free amino acids (especially arginine) concentrations in new and middle-aged needles was also greater than in old needles. This indicate that free amino acids in new and middle-aged needles may be more sensitive to N deposition compared to old needles. Moreover, concentrations of total free amino acids, arginine, histidine, gamma-aminobutyric acid and alanine in middle-aged needles exhibited a strong relationship with N deposition (P < 0.05). Needle delta N-15 values showed a strong gradient from central Guiyang to the rural area, with more positive delta N-15 (especially in old needles) in the city center (0-5 km) and more negative delta N-15 (especially in old needles) in rural area (30-35 km). These suggest that N deposition in the urban center may be dominated by N-15-enriched NOx-N from traffic exhausts, while it is dominated by isotopically light atmospheric NHx-N from agriculture in rural area. Soil delta N-15 decreased slightly with distance from the city center, and the difference in delta N-15 values between the soil and needles (especially for old needles) increased significantly with the distance gradient, indicating that atmospheric N deposition may be an important N source for needles. This study provides novel evidence that free amino acids in needles and age-dependent needle delta N-15 values are useful indicators of atmospheric N deposition.