Technical Abstract: Pyrrolizidine alkaloid-containing plants are found throughout the world and are common in the genus Senecio. Many pyrrolizidine alkaloids are toxic and cause poisoning in livestock and in humans (Bull et al., 1968; Johnson et al., 1989; Mattocks, 1986; Prakash et al., 1999; Stegelmeier et al., 1999). The alkaloid types and concentrations vary between plant species. In addition, within a species of plant, concentrations vary with environment and location. Most pyrrolizidine alkaloid-containing plants produce mixtures of the free bases and their corresponding N-oxides in varying concentrations. In general, the free base and N-oxide forms are similar in toxicity if absorbed via the gut (Mattocks, 1986; Molyneux et al., 1991). Rapid, sensitive and specific diagnostic techniques are needed to determine the plants and conditions under which livestock are likely to be poisoned , and to monitor feeds and food supplies that are intended for livestock or human consumption. In this study a competitive inhibition enzyme-linked immunosorbent assay (CI-ELISA) for riddelliine, riddelliine N-oxide and other closely related pyrrolizidine alkaloids was developed. This assay allowed us to estimate the total pyrrolizidine alkaloid content in Senecio riddellii and Senecio madagascarensis plant material and in Senecio riddellii admixtures with alfalfa.