Experimental conditions for the small scale alkylation of the alpha-amino group of the opioid peptide methionine-enkephalin using the method of reductive alkylation are described. The generality of the method is established by describing in detail the synthesis, purification, and structure elucidation of the N alpha-ethyl, isopropyl, phenethyl, and cyclopropylmethyl derivatives of methionine-enkephalin. By a combination of amino acid and mass spectral analysis it is possible to carry out the structure analysis of nanomole quantities of these modified peptides, making direct chemical modification and structure elucidation of carrier-free tritiated and radioiodinated enkephalins feasible. Using these chemical procedures, a homologous series of N alpha-n-alkyl derivatives of methionine-enkephalin has has been synthesized. It is shown that the relative binding and opiate activity of these N alpha-alkyl derivatives, using several standard in vitro assay systems, depends on the tissue source used. The results of this study are analyzed by considering recent reports indicating the existence of multiple opiate receptors.