A series of puromycin analogues, 3'-N'-(S-substituted L-cysteinyl)puromycin aminonuleosides, has been preepared and examined as substrates for ribosomal peptidyl transferase. S-Substituted N-tert-butyloxycarbonyl-L-cysteines were coupled with puromycin aminonucleoside using dicyclohexylcarbodiimide and N-hydroxysuccinimide. Removal of the t-Boc blocking group with anhydrous trifluoroacetic acid gave the desired puromycin analogues. Kinetic studies indicate that the nonaromatic aminoacyl analogues of puromycin are effective substrates for the peptidyl transferase reaction. In addition, the discovery of the existence of hydrophilic character beyond the region normally occupied by hydrophobic amino acid R groups of the aminoacyladenyl termini of tRNA molecules, and the proper exploitation of this information, has provided the first active purmoycin analogue possessing a hydrophilic amino acid.