A spin-labeled penicillin derivative has been synthesized by acylation of the amino nitrogen of 6-aminopenicillanic acid with the nitroxyl spin-label 2,2,5,5-tetramethyl-1-oxypyrroline-3-carboxylic acid and has been characterized by chemical methods. With Bacillus cereus beta-lactamase I, the steady-state kinetic parameters k(cat) approximate to 1810 s(-1) and K-M approximate to 118 x 10(-6) M at 22 degrees C and pH 7 showed that this paramagnetic substrate probe is as kinetically specific and catalytically reactive as is the classical substrate benzylpenicillin. From analysis of electron nuclear double resonance (ENDOR) spectra, the principal hyperfine coupling (hfc) components of specific protons in the fused beta-lactam and thiazolidine ring were determined. The dipolar hfc components yielded electron-proton separations between the unpaired electron on the nitroxyl group and protons in the penicillin moiety. The conformation of the spin-labeled penicillin was determined on the basis of torsion angle search calculations constrained by ENDOR-determined distances. The ENDOR-assigned conformation of the spin-labeled penicillin is almost identical to the X-ray-defined structure of amoxycillin (Boles, M. O.; et al. Acta Crystallogr., Sect. B 1978, 34, 461-466). We have also determined by ENDOR the location of a methanol molecule that is hydrogen-bonded to the amide NH group, coinciding almost exactly with the position of a similarly hydrogen-bonded water molecule in the X-ray structure of amoxycillin. The location of the hydroxyl group of the methanol molecule, restricted to the "endo" side of the beta-lactam ring on the basis of ENDOR distance constraints, appears as if it were poised for nucleophilic attack on the carbonyl carbon of the beta-lactam. This observation is important with respect to the mechanism of hydrolysis of beta-lactams because "endo" attack of a fused beta-lactam compared to "exo" attack is considered to be hindered sterically but favored stereoelectronically.