Improved symbiosis establishment between effective rhizobia and forage legumes is often important for maximizing sward yields, particularly when seeding is done on acidic, low-fertility soils. New information regarding the earliest events in this process could provide insights into the development of germplasm, both plant and bacterial, better adapted to such soils. A recently developed plate model system containing a narrow range of limed (pH 4.71–4.99), nonsterile soils (see article by Staley in Soil Science 167, pp. 211–221) was employed to investigate initial nodulation of white clover (Trifolium repensL.) seedlings. Our objectives were to further evaluate nodulation assessment methodology (staining and nodule stage), distinguish between soil pH-induced plant and rhizobia limitations on nodulation by employing (daily) saturation inoculation, and determine if previously observed nodulation reduction by low pH soil was a consequence of inhibition of symbiosis establishment or nodule maturation by utilizing agusA-marked mutant ofRhizobium leguminosarumbv.trifolii.Over the course of all experiments in all soils, rhizobial MPN populations never declined below 1.3 × 104CFU g−1dry soil. Significant (P≤ 0.05) nodulation responses to soil pH increase were found for both the wildtype (gus−) and the mutant (gus+) stains, whether assessed using unstained (fresh) or stained (methylene blue or GUS) roots. The pattern ofgusAnodule ontogeny under saturation levels of rhizobia, relative to soil pH, suggests that inhibition of symbiosis establishment (cellular effects), rather than differences in nodule maturation (tissue effects), is the explanation for our observed soil pH effects on nodulation. Re-investigation of root responses revealed small but significant (P≤ 0.05) differences, due to soil pH, primarily for lateral roots. Taken together, these results corroborate our previous, wildtype rhizobia findings of a positive effect of soil low-level liming (only 0.25 pH increase) on nodulation and extend them to earlier times (4–6 DAP) in symbiosis establishment, but they refute our previous finding of no root growth and developmental effects. More importantly, they suggest that nodulation reduction in the lower pH soils of our model system was caused by disruption of rhizobia function (but not viable populations) or root hair growth and/or function.