Purpuromycin (1) is a natural antibiotic with a broad spectrum of activity encompassing bacteria, fungi and protozoa. A new series of derivatives of 1 was prepared by the modification or replacement of the C-4 hydroxyl group. The physico-chemical characteristics and the in vitro antimicrobial activity of these new semisynthetic purpuromycin derivatives are reported. Attachment of a variety of bulky groups to the C-4 hydroxyl group as well as acylation or mesylation of 1 gave derivatives with significantly reduced antifungal activity, while the antimicrobial activity of these derivatives against Gram-positive and Gram-negative bacteria was only slightly decreased. All compounds were inactive against Escherichia coli. The C-4 epimers showed different in vitro activity as compared with those having the natural configuration, particularly against fungi.