A set of 58 nuclearly encoded thylakoid-integral membrane proteins from four plant species was identified, and their amino termini were assigned unequivocally based upon mass spectrometry of intact proteins and peptide fragments. The dataset was used to challenge the Web tools ChloroP, TargetP, SignalP, PSORT, Predotar, and MitoProt II for predicting organelle targeting and transit peptide proteolysis sites. ChloroP and TargetP reliably predicted chloroplast targeting but only reliably predicted transit peptide cleavage sites for soluble proteins targeted to the stroma. SignalP (eukaryote settings) accurately predicted the transit peptide cleavage site for soluble proteins targeted to the lumen. SignalP (Gram-negative bacteria settings) reliably predicted peptide cleavage of integral thylakoid proteins inserted into the membrane via the "spontaneous" pathway. The processing sites of more common thylakoid-integral proteins inserted by the signal recognition peptide-dependent pathway were not well predicted by any of the programs. The results suggest the presence of a second thylakoid processing protease that recognizes the transit peptide of integral proteins inserted via the spontaneous mechanism and that this mechanism may be related to the secretory mechanism of Gram-negative bacteria.