Bacterial group IIC introns are a subclass of group II intron ribozymes that are typically located downstream from transcriptional terminators. Class IIC-attC introns constitute a monophyletic subset of subgroup IIC, which preferentially insert into site-specific recombination sequences for integron integrases (attC). attCs are a diverse family of nucleotide sequences composed of conserved inverted repeats that flank a variable, but palindromic, central region. In this study, we used both PCR and colony patch hybridization methods to determine the basis for recognition of the attC(aadA1) stem-loop motif by the Serratia marcescens intron (S.ma.I2) in vivo. The quantitative results showed that mobility into the wild-type site occurs at a frequency of 18%, and is strongly biased by the orientation of the homing site relative to the direction of DNA replication. S.ma.I2 mobility results into mutant attC(aadA1) sites are consistent with recognition of stem-loop motifs in unwound DNA. The homing frequency results showed that, while the entire attC sequence is not necessary for recognition of the insertion site, short deletions of the attC stem-loop motif inhibited the intron mobility. Moreover, our data show that S.ma.I2 requires a bulged base in the folded attC stem for high homing frequency. We demonstrate that the IBS1/IBS3 motifs and two bulge bases conserved among attCs determine S.ma.I2 homing specificity for the attC bottom strand. These results suggest that class IIC-attC introns tolerate attC variation by recognition of a bulged hairpin DNA motif rather than a specific sequence.