The biochemical properties of tetrahydrofuranyl (THF) carbapenems, carbapenems with THF substituents, were evaluated with respect to enzyme stability, binding to penicillin-binding proteins (PBPs), and penetration into gram-negative organisms. THF carbapenems showed increased stability to hog renal dehydropeptidases (DHPs) compared to that of imipenem or meropenem and were more stable to human DHP than imipenem (<10% hydrolysis compared to that for imipenem). THF carbapenems were stable to hydrolysis by all serine β-lactamases tested. CL 191,121, a prototype THF carbapenem, was more stable to hydrolysis by carbapenem-hydrolyzing serine β-lactamases such as IMI-1 and Sme-1 than imipenem, with a relative kcat value of <20% for imipenem. Similar to imipenem and meropenem, THF carbapenems were not stable to the metallo β-lactamases CcrA and L1. However, CL 191,121 bound to all Staphylococcus aureus PBPs at concentrations that were less than or equal to the MICs. The THF carbapenems bound to PBPs from Escherichia coli and Pseudomonas aeruginosa, with the highest affinities being for PBPs 2 and 4, as noted with imipenem. The affinities for PBPs 1a and 1b in E. coli were reduced for the THF carbapenems compared to that for imipenem, even though the MICs of the THF carbapenems for E. coli strains were lower than those of imipenem. The penetrability of the THF carbapenems into Serratia marcescens S6, which produces the Sme-1 carbapenem-hydrolyzing β-lactamase, was 2.4 to 7.8 times less than that of imipenem. Compounds CL 190,294 and CL 188,624 showed good penetrability, with permeability coefficient values comparable to those of the rapidly penetrating agents cephaloridine, imipenem, meropenem, and biapenem. Decreased penetration into wild-type P. aeruginosa was suggested by the high MICs of the THF carbapenems (MICs, 16 to 32 μg/ml), despite equivalent or better binding to P. aeruginosa PBPs than that of imipenem. However, the MICs of the THF carbapenems for wild-type P. aeruginosa compared to that for an OprD2 mutant generally varied no more than 2-fold, but those of imipenem and other carbapenems differed 16-fold. These data indicated that THF carbapenems do not appear to enter through protein OprD2. In conclusion, the THF carbapenems exhibited stability to hydrolysis by renal DHPs and serine β-lactamases, exhibited strong binding to essential PBPs from E. coli and S. aureus, and penetrated gram-negative enteric bacteria at rates comparable to those for meropenem and biapenem.