Inverted sequences of the chloramphenicol acetyltransferase (CAT) reporter gene were fused to a soybean tRNA(met(i)) gene lacking a terminator such that the tRNA(met(i)) sequences caused the co-transcription of CAT antisense sequences by RNA polymerase III. When electroporated into carrot protoplasts, these antisense DNA constructs suppressed CAT enzyme activity expressed from co-electroporated DNAs containing the CAT gene downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. Our most effective construct, an antisense sequence complementary to the 3' portion of the CAT gene, inhibited CAT activity five-fold greater than an antisense construct expressed by RNA polymerase II from the cauliflower mosaic virus 35S RNA promoter. These results indicate that antisense sequences transcribed by RNA polymerase III should efficiently suppress gene expression in plants.