Partial amino acid sequences of two mitochondrial cytochrome P-450's, P-450 (SCC) and P-450 (11 beta), and two microsomal cytochrome P-450's, P-450 (C-21), and P-450 (17 alpha, Lyase), from adrenal cortex were analyzed and compared. Mitochondrial P-450's and microsomal P-450's were different in the amino acid sequences at their NH2-terminals. The sequences of microsomal P-450's started from terminal methionine and were highly hydrophobic, whereas those of mitochondrial P-450's lacked NH2-terminal methionine and were not hydrophobic. These findings strongly suggest that the NH2-terminal portions of newly synthesized P-450's determine their intracellular localization to different cell organelles.