Determinative and confirmatory methods of analysis for pirlimycin (I) residue in bovine milk and liver have been developed based on HPLC-thermospray (TSP) MS. Milk sample preparation consisted of precipitating the mill proteins with acidified acetonitrile followed by a solvent partitioning with a mixture of n-butyl chloride and hexane extraction of I from the aqueous phase into methylene chloride (MC), and solid-phase extraction clean-up. For liver, samples (2 g) were extracted with 0.25% trifluoroacetic acid in acetonitrile. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, then extracted with MC. The MC was evaporated to dryness and the dried residue reconstituted in 2.0 ml of 0.1 M ammonium acetate for analysis. A chromatographically resolved stereoisomer of I with TSP-MS response characteristics identical to I was used as an internal standard (I.S.) for quantitative analysis based on the ratio of peak areas of I to I.S. in the protonated molecular-ion chromatogram at m/z 411.2. The method for milk was validated by the analysis of control milk samples spiked with I at concentrations from 0.05 to 0.8 micrograms/ml. The overall recovery of pirlimycin across this concentration range was 95.4% +/- 8.7%. The limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were validated to be 0.05 micrograms/ml and 0.10 micrograms/ml, respectively. The method for liver was validated by the analysis of control liver samples spiked with I at concentrations ranging from 0.025 to 1.0 micrograms/g. The overall recovery of pirlimycin was 97.6% +/- 5.1% in this concentration range. The validated limit of quantitation (LOQ) and limit of confirmation (LOC) of the method were 0.025 micrograms/g and 0.10 micrograms/g, respectively. Four diagnostic ions for I were monitored for confirmation: the pseudo-molecular ions (M+H)+ at m/z 411.2 (35Cl) and m/z 413.2 (37Cl), and fragment ions at m/z 375.2 and 158.1. Confirmatory criteria were defined for these assays.