We demonstrate that naturally occurring C₁₄ and C₁₆-specific acyl-acyl carrier protein (ACP) desaturases from plants can complement the unsaturated fatty acid (UFA) auxotrophy of an Escherichia coli fabA/fadR mutant. Under the same growth conditions, C₁₈-specific Δ⁹-stearoyl (18:0)-ACP desaturases are unable to complement the UFA auxotrophy. This difference most likely results from the presence of sufficient substrate pools of C₁₄ and C₁₆ acyl-ACPs but a relative lack of C₁₈ acyl-ACP pools in E. coli to support the activities of the plant fatty acid desaturase. Based on this, a substrate-dependent selection system was devised with the use of the E. coli UFA auxotroph to isolate mutants of the castor Δ⁹-18:0-ACP desaturase that display enhanced specificity for C₁₄ and C₁₆ acyl-ACPs. Using this selection system, a number of desaturase variants with altered substrate specificities were isolated from pools of randomized mutants. These included several G188L mutant isolates, which displayed a 15-fold increase in specific activity with 16:0-ACP relative to the wild-type castor Δ⁹-18:0-ACP desaturase. Expression of this mutant in Arabidopsis thaliana resulted in the accumulation of unusual monounsaturated fatty acids to amounts of >25% of the seed oil. The bacterial selection system described here thus provides a rapid means of isolating variant fatty acid desaturase activities for modification of seed oil composition.