We previously reported that aminopeptidases, alkaline phosphatase, esterase, and glycosidases are present on the surface of mammalian cells including macrophages and lymphocytes, and their specific inhibitors (e.g., bestatin, amastatin, forphenicine, esterastin, ebelactones A and B) produced by Actinomycetes modify the functions of immunoresponsive cells. We further continued screening for inhibitors of aminopeptidase B and discovered arphamenines A and B, which are completely specific inhibitors of aminopeptidase B and enhance immune responses. This paper reports the isolation and characterization of arphamenines. The producing strain is Chromobacterium violaceum BMG361-CF4. Arphamenines were isolated from 9 liters of culture filtrate via Amberlite XAD-4 adsorption, CM-Sephadex C-25 chromatography with a linear NaCl gradient, and Sephadex LH-20 chromatography, yielding purified arphamenine A (92 mg, IC50 0.006 μg/ml for aminopeptidase B) and arphamenine B (32 mg, IC50 0.002 μg/ml for aminopeptidase B). Arphamenine A has a molecular formula of C18H24N4O3 (m/z 321 [M+1] by FD mass spectrometry), melts at 117-119°C (dec.), and has a specific rotation [α]D²⁰ of +46° (c 0.3, 0.1 N HCl). Arphamenine B has a molecular formula of C18H24N4O4 (m/z 337 [M+1] by FD mass spectrometry), melts at 117-119°C (dec.), and has a specific rotation [α]D²⁰ of +49° (c 0.3, 0.1 N HCl). Both are soluble in water and methanol but insoluble in butanol, chloroform, and ether, and give positive reactions with ninhydrin, Rydon-Smith, Sakaguchi, and 2,3,5-triphenyltetrazolium chloride reagents. Their structures (determined in subsequent papers) are 5-amino-8-guanidino-4-oxo-2-phenylmethyloctanoic acid (arphamenine A) and 5-amino-8-guanidino-2-(4-hydroxyphenylmethyl)-4-oxooctanoic acid (arphamenine B). Arphamenines A and B strongly inhibit aminopeptidase B (IC50 0.006 and 0.002 μg/ml, respectively) but show no inhibition of aminopeptidase A or leucine aminopeptidase even at concentrations >100 μg/ml. Oral administration of 0.005-5.0 μg/mouse of arphamenines A and B augments delayed-type hypersensitivity to sheep red blood cells in CDF1 mice older than 10 weeks. They have low toxicity (no death after intraperitoneal injection of 500 mg/kg to mice), no antimicrobial activity at 100 μg/ml, and inhibit sarcoma 180, IMC carcinoma, and the generation of suppressor cells.