We have compared RNA polymerase promoter activities in PCR-generated DNA fragments for use in the in vitro transcription of cRNA probes. Sense oligonucleotide primers, specific for the mouse acidic fibroblast growth factor gene, were synthesized with 5' extensions containing promoter sequences for the T7, T3 and SP6 RNA polymerase promoters. A common antisense primer was used with each of the promoter/aFGF primers to prepare PCR-generated DNA fragments (minigenes). In vitro transcription efficiency for each of these constructs was evaluated by incorporation of radioactivity into the cRNA products. We find that both the T7 and T3 promoters can direct the synthesis of cRNA probes of high specific activity from a PCR-generated DNA fragment, but that SP6 cannot. No detectable cRNA product was obtained using either T7 polymerase on the T3/minigene or T3 on the T7/minigene. Antisense cRNA probes, transcribed from minigene constructs were used for both Northern and in situ hybridization studies. A PCR-generated DNA fragment with RNA polymerase promoter sequences at each end provides a single template for synthesis in vitro of either sense or antisense cRNA probes.