Carpetimycins A and B, two new carbapenem β-lactam antibiotics related to thienamycin, olivanic acid derivatives and PS-5, were isolated from the culture filtrate of Streptomyces sp. KC-6643. The strain was first cultured in Erlenmeyer flasks (100 ml medium containing 3.6% starch, 2.2% soybean meal, 1.5% cotton seed oil, 0.62% Na₂HPO₄•12H₂O, 0.1% KH₂PO₄, 0.05% MgSO₄•7H₂O, 0.001% FeSO₄•7H₂O and 0.0005% CoCl₂•6H₂O, 29°C, 72 h) and then scaled up to a 200-liter fermentor (29°C, aeration 100 liters/min, agitation 240 rpm, inner pressure 0.5 kg/cm², 144 h). The culture broth was filtered with Dicalite, and the antibiotics were purified via successive steps: adsorption on Diaion PA-306, elution with 20% NaCl, desalting on Diaion HP-20, separation on Amberlite IRA-458 (carpetimycin A eluted with 0.9% NaCl, carpetimycin B with 20% NaCl), followed by column chromatography (QAE-Sephadex A-25, Diaion HP-20, Sephadex G-10 for A; DEAE-Sephadex A-25, Diaion HP-20, Sephadex G-10 for B) and HPLC (Bondapak C18/Porasil B) purification. Finally, pure carpetimycin A (39 mg as sodium salt) and B (210 mg as disodium salt) were obtained. Carpetimycin A sodium salt is a colorless solid melting above 145°C with decomposition, [α]²⁴ᴰ -27° (c 1.7, water), showing UV (H₂O) maxima at 240 nm (E¹%₁cm 369) and 288 nm (E¹%₁cm 300), IR (KBr) peaks at 1770, 1695, 1625 and 1265 cm⁻¹, ¹H-NMR (D₂O) signals (δ 1.83, 1.91, 2.65, 3.54, 4.32, 4.43, 4.98, 6.92, 8.10), and a molecular formula of C₁₄H₁₈N₂O₈S (consistent with mass, ¹³C-NMR and analytical data). Carpetimycin B disodium salt is a colorless solid melting above 130°C with decomposition, [α]²⁴ᴰ -145° (c 1, water), exhibiting UV (H₂O) maxima at 240 nm (E¹%₁cm 357) and 285 nm (E¹%₁cm 305), IR (KBr) peaks at 1770, 1695, 1625, 1270-1220 and 1050 cm⁻¹, ¹H-NMR (D₂O) signals (δ 2.15, 2.23, 2.65, 3.62, 4.42, 4.50, 5.04, 6.95, 8.13), and a molecular formula of C₁₄H₁₈N₂O₉S₂. Antibacterial activity assays (Brain Heart Infusion agar, 10⁶ cells/ml) revealed strong activity of both compounds against Gram-positive (e.g., Staphylococcus aureus 209P JC-1, MIC 0.39 µg/ml for A; Bacillus subtilis ATCC6633, MIC 0.2 µg/ml for A) and Gram-negative bacteria (e.g., Escherichia coli NIHJ JC-2, MIC 0.05 µg/ml for A; Pseudomonas aeruginosa NCTC10490, MIC 6.25 µg/ml for A), including β-lactamase-producing strains (e.g., Serratia marcescens 4*, MIC 3.13 µg/ml for A). Carpetimycin A was 8-32 times more active than B. Both antibiotics inhibited β-lactamases: carpetimycin A had I₅₀ values of 0.64 ng/ml (against E. coli ML 1410 R EC-1 penicillinase, substrate PCG) and 0.52 ng/ml (against P. vulgaris 69 cephalosporinase, substrate CER), while carpetimycin B had I₅₀ values of 0.21 ng/ml and 0.74 ng/ml, respectively. The structures of carpetimycins A and B, including their stereochemistry, were elucidated by spectral data and conversion of B to A, with final confirmation via X-ray crystallographic analysis of the p-bromobenzyl ester of carpetimycin A.