Membrane properties of fatty acyl coenzyme A (CoA) and acylcarnitine have been studied with 1-pyrenebutyryl-CoA and (1-pyrenebutyryl) carnitine. These molecules have a spectroscopic properties of pyrene and its derivatives and exhibit biological and chemical characteristics related to acyl esters. (1-Pyrenebutyryl) carnitine (PBC) is more soluble in nonpolar solvents than 1-pyrenebutyryl-CoA (PB-CoA), and critical micelle concentrations of both compounds resemble the medium-chain fatty acyl esters. PB-CoA inhibits phosphorylating (ADP-stimulated) respiration in rat liver mitochondria noncompetitively (KI = 2 microM) and carnitine palmitoyl-CoA and octanoyl-CoA transferases competitively (KI = 2.1 microM and 15 microM, respectively). PBC does not inhibit carnitine palmitoyl-CoA and transferase or mitochondrial respiration when glutamate-malate or succinate (+ rotenone) is used as the respiratory substrate. PBC is a potent inhibitor of phosphorylating respiration with either palmitoylcarnitine (I50 = 1.4 microM) or octanoylcarnitine (I50 = 40 nM) as the respiratory substrate. The mitochondrial carnitine-acyl-carnitine translocase is competitively inhibited by PBC with a KI = 0.6 microM for palmitoylcarnitine exchange and 23 nM for carnitine exchange. PBC and PB-CoA exhibit excimer and monomer fluorescence, the relative intensities of which are functions of their microscopic concentrations. PB-CoA is accessible only to the outer half of artificial lipid vesicles while PBC may cross lipid vesicle bilayers. PBC in the inner half of the bilayer appears "trapped" i.e., not easily removed by exogenous bovine serum albumin, which binds PBC.