The major alternative polyadenylation sites in the Chinese hamster dihydrofolate reductase (dhfr) gene have been identified by DNA sequencing and RNase protection experiments. Comparison of the 3' gene sequence and polyadenylation sites with those of the mouse reveals that, despite an overall sequence homology, the major sites are different in the two species. A series of minigenes was constructed containing the dhfr promoter and the first intron but lacking the four large introns of the genomic sequence. These minigenes contained either all three polyadenylation sites, no polyadenylation sites, or just the first site. All of these minigenes, as well as a cosmid clone containing the full genomic sequence, could transform DHFR-deficient Chinese hamster ovary cell mutants to a DHFR-positive phenotype with approximately equal efficiencies. A minigene lacking the first intron was markedly less efficient. Analysis of dhfr mRNA from transfectant clones derived from minigenes showed that the dhfr polyadenylation sites were used when included, but novel sites were often used in addition. When endogenous polyadenylation sites were absent, new sites in flanking carrier or host DNA were recruited. Transfectants produced by the full genomic dhfr gene yielded mRNA species that were identical in size and relative abundance to the endogenous dhfr gene. The results indicate that the minimal signals for polyadenylation are not complex and can be easily acquired from foreign sequences.