The influence of water stress on toxic alkaloid concentrations in threadleaf groundsel (Senecio longilobus Benth.) was investigated in a controlled environment by monitoring leaf xylem water potential during a 32-day period, and then analyzing for alkaloid concentration by plant part. Total plant alkaloid concentration increased with increasing severity of water stress. The most severe water stress (−2.0 MPa) for the 32-day period increased alkaloid concentration by 4.6 times, compared to the least stress imposition (−1.0 MPa). Averaged across watering regimes, alkaloid concentrations (percent dry weight) were highest in threadleaf groundsel roots (0.088%) and occurred in similar concentrations in stems (0.046%) and leaves (0.050%). These results indicate the importance of considering the influence of plant water stress when investigating secondary plant compounds.