The purpose of this research was twofold, a) to directly demonstrate import in vivo of a native plant peroxisomal protein into peroxisomes of transiently transfected mammalian cells, and b) to identify the targeting signal and amino acid substitutions thereof which preserve translocation of this plant protein into these peroxisomes. The protein selected for study was cottonseed isocitrate lyase (ICL), a glyoxylate cycle enzyme which participates in storage oil mobilization in oilseed cotyledons. Cultured mammalian cells were selected as the import system because of previous success by others with transient transfections and import of heterologous (not plant, however) proteins, and because neither a plant in vitro or transient in vivo import system was established. Optimized transient transfections of cultured CV-1 monkey kidney, mouse L, HeLa, and CHO cells resulted in punctate, anticottonseed-ICL-dependent immunofluorescent patterns. Colocalization in a CVH Px110 cell line of ICL with either endogenous catalase or with stably expressed CAT-PMP20/AKL (chloramphenicol acetyltransferase with a C-terminal-appended 12 amino acids ending with Ala-Lys-Leu) demonstrated targeting of ICL to peroxisomes. Direct evidence for translocation of ICL into CHO cell peroxisomes was obtained from digitonin permeabilization experiments. The necessity of the C-terminal tetrapeptide, KARM-COOH, was demonstrated in CHO and CV-1 cells when removal of this tetrapetide (leaving ICL-VVA-COOH) abolished import into peroxisomes. This result is in general agreement with Olsen et al. (The Plant Cell 5, 941-952 (1993)) who demonstrated that the 37 C-terminal amino acids of oilseed rape ICL were necessary for import in vivo in transgenic plants. The findings of Behari and Baker (J. Biol. Chem. 268, 7315-7322 (1993)), however, indicate that the C-terminal portion of castor bean ICL is dispensible for import in vitro. Single or multiple conservative amino acid substitutions at each position of the C-terminal tripeptide of native cottonseed ICL (S for A, K for R, L for M, SK for AR, SKL for ARM) preserved import of the enzyme in vivo into CHO cell peroxisomes. The demonstrated targeting and translocation of plant ICL and C-terminal modifications thereof into mammalian cell peroxisomes provide important additional evidence for evolutionary conservation of peroxisome import machinery, especially relative to the PTS1 sequence.