The biological activity of 14 analogues of sparsomycin (1) was studied in cell-free systems of Escherichia coli, Saccharomyces cerevisiae, and Sulfolobus solfataricus by measuring the inhibition of protein synthesis. The inhibition of L1210 colony formation in soft agar and bacterial cell growth in solid as well as in liquid medium was also examined. Each analogue possesses not more than two structural modifications of the sparsomycin molecule. This enabled us to determine unambiguously several structural and stereochemical features that are required for an optimal biological activity in these assays. Sparsomycin, having the SCRS chirality, is the most potent of the four possible stereoisomers. The results obtained with compounds 5-7 indicate that the presence of an oxygen atom on the S (alpha) atom is essential. Substitution of the bivalent sulfur atom by a CH2 group (10) or of the SCH3 moiety by a Cl atom (12) affects the activity of the molecule partially. Compound 12 is surprisingly active against intact cells. Substitution of the C(6)-CH3 group by a H(14) reduces the activity of the molecule. Isomerization of the trans double bond into the cis double bond yields cis-sparsomycin (15), which is inactive. The hydrophobic derivatives 8, 9, and 11 are considerably more active than sparsomycin; thus the ribosomal binding site for sparsomycin may have a hydrophobic character.