Myosin subfragment-1 was modified with pyridoxal-5'-phosphate, a lysine modifying agent. Approximately two lysines could be blocked with a concomitant decrease in the actin-activated Mg2(+)-ATP-ase activities of S-1 remained unchanged. This selective inhibition of actin-activated Mg2(+)-ATP-ase activity of S-1 by pyridoxal-5'-phosphate was further characterized by kinetic studies. The double reciprocal plot revealed no change in the Vmax, while KM increased from 15 microM to 36 microM indicating that the modification reduced the actin affinity of S-1. The effects of pyridoxylation of S-1 were compared to those of 2, 4, 6 trinitrobenzene-sulfonate modification of S-1. The two types of lysine modification are strikingly different. The reactive lysine residue (Lys 83) remained unmodified after pyridoxylation of S-1 thus the effects of trinitrophenylation could be revealed independently in the double-modified sample. Furthermore after trinitrophenylation the S-1 fragments were found to be protected against partial tryptic digestion in the presence of nucleotides.