Pseudomonas, due to its diversity in habitat and metabolic type, makes it have broad prospects applying in bioremediation, bioconversion, and biocontrol, while the introduction of exogenous gene is the key link to genetically modified Pseudomonas. The preparation and transformation of competent cells are the important methodological basis of the introduction of exogenous gene. In this paper, three Pseudomonas strains (P. putida TS11, P. stutzeri DNB, and P. mendocina JJ12) isolated from a petroleum-contaminated soil were taken as the recipient strains, and a three-factor and four-level orthogonal experiment was conducted to investigate the effects of CaCl2 concentration, heat shock duration, and recovery duration on the preparation and transformation efficiency of the strains competent cells. The results showed that CaCl2 concentration was the most important factor affecting the transformation efficiency (P<0.05), and the transformation efficiency was improved markedly when the Pseudomonas cells were repeatedly washed with sterile distilled water before the preparation of competent cells. When the P. putida TS11 cells were treated with 100 mmol L-1 of CaC12, heat-shocked for 3 minutes at 42 degrees, and incubated for 1.5 hours at 30 degrees C, the P. stutzeri DNB cells were treated with 50 mmol . L-1 of CaCl2, heat-shocked for 6 minutes, and incubated for 1.5 hours, and the P. mendocina JJ12 cells were treated with 75 mmol . L-1 of CaCl2, heat-shocked for 4. 5 minutes, and incubated for 0. 5 hours, the transformation efficiency of exogenous plasmids in the three strains all achieved 10(5) cells . microg-1 DNA.