Abstract The antibacterial potencies of 2a and 4 are shown to be diminished considerably from their penam analogues, penicillin G ( 1a) and mecillinam ( 3). Despite this, 2a is a substrate for bacterial β-lactamases, and compounds 6a, 8 and 10 were found to be β-lactamase inhibitors. Penicillin-binding protein (PBP) studies indicate that penicillin G and mecillinam have much greater affinity for these enzymes than the (2,3)-β-methylenepenam analogues. Based on a comparison of hydrolytic stabilities, it is proposed that the change in biological properties is due to conformational differences between the two types of penam nuclei. The cyclopropyl methylene of 2a and 4 blocks the side chain from forming an oxazolone with the β-lactam carbonyl. Hence, activation of the β-lactam is prevented and the molecules are rendered less active. We thus conclude that 19 is the biologically active conformation of penicillin antibacterials, and further suggest that the interaction of such antibiotics with their bacterial enzyme targets involves intermediates such as 25– 27.