Abstract. Onduso SO, Ng‘ang‘a MM, Wanjohi W, Hassanali A. 2017. Determination of pyrrolizidine alkaloids levels in Symphytum asperum. Biofarmasi J Nat Prod Biochem 15: 65-73. The levels of the pyrrolizidine alkaloids (PAs) in the roots and leaves of comfrey (Symphytum asperum Lepech) from two agro-ecological zones in Kenya, Kiambu, and Kisii counties during wet (April 2012) and the dry (September 2012) seasons were determined in the present study. The samples were dried beneath the shade for four weeks, continued with ground and extraction of the PAs by applying different solvent systems. The result displayed no significant difference in the percentage of dry weights for the leaves and the roots samples collected from Kisii and Kiambu counties during the wet and dry seasons (p>0.05 at 95 % confidence limit). Gas Chromatography (GC) combined with a Mass spectrometer (MS) was used to analyze the samples and characterize and quantify major components. Among four PAs identified in the root samples (echimidine (7), 7-acetyllycopsamine (17), 3‘-acetyllycopsamine (18), triangularine (19), and heliosupine (24)), a significant amount of 7-Acetyllycopsamine (17) was evident during the wet season as compared to the dry season (p=0.033, ?=0.05, t-test). The other pyrrolizidine alkaloids measured between the two seasons did not vary significantly (p>0.05, ?=0.05, t-test). All the compounds reported in the root samples are associated with toxicity, and their values exceed the tolerable levels recommended by relevant regulatory bodies in various countries. One of the most toxic PAs, echimidine (7), was found in all the root samples. However, the leaf samples showed PAs level below detectable limits using GC-MS. Further study is needed to examine whether PAs are produced from the leaves collected at different maturation levels and from a broader plant profile. Furthermore, it is necessary to know the levels of a particular PA and related analogs in a larger profile of the plants cultivated in other agroecological zones in Kenya. A study on the toxicity of heliosupine (24) is of future interest since it exists in significant amounts, yet such studies have not been done on this constituent. Also, preparative isolation of the PAs should be performed to provide reference standards in other quantification facilities that utilize Gas Chromatography because GC-MS is a rather expensive method.