A variety of potential inhibitors of de novo fatty acid biosynthesis have been tested for activity in Acholeplasma laidlawii B. Two compounds, avidin and N,N-dimethyl-4-oxo-2trans-dodecenamide (CM-55), an antimicrobial fatty amide, strongly inhibit de novo biosynthesis without nonspecific toxic effects at moderate dosages. Avidin is the more potent inhibitor, abolishing de novo fatty acid synthesis and greatly reducing the chain elongation of exogenous fatty acids at level of 25 U/l. CM-55 gives complete inhibition of de novo biosynthesis only at low temperatures and inhibits exogenous fatty acid elongation to a variable extent. However, CM-55 is still a more potent antilipogenic agent in this organism than is the fungal antibiotic cerulenin. Cells cultured with avidin grow only when one or more exogenous medium- or long-chain fatty acids are added to the growth medium. The extent of cell growth under these conditions depends primarily on the physical properties of the exogenous fatty acid(s). In general, fatty acids giving diacylglycerolipids of very high or very low fluidity are unsuitable growth substrates, while those whose diacylglycerol derivatives are of intermediate fluidity support fair to good cell growth.