Multicellular spheroids composed of transformed cells are known to mimic the growth characteristics of tumors and to develop gradients in proliferation with increasing size. This progressive accumulation of quiescent cells is presumably an active process that occurs in response to the microenvironmental stresses that develop within the three-dimensional structure, and, yet, little is known regarding either the signals that induce the cell cycle arrest or the molecular basis for the halt in proliferation. We have previously reported that regulation of cyclin-dependent kinase (CDK) inhibitors (CKIs) differs in monolayer versus spheroid cell culture. In this study, we have examined the expression of three CKIs in EMT6 mouse mammary carcinoma and MEL28 human melanoma spheroids, as a function both of spheroid size and of location within the spheroid. We report that expression of the CKIs p18(INK4c), p21(waf1/cip1), and p27(Kip1) all increase as the spheroid grows and develops a quiescent cell fraction. However, by examining protein expression in discrete regions of the spheroid, we have found that only p18(INK4c) and p27(Kip1) expression positively correlate with growth arrest, whereas p21(waf1/cip1) is expressed predominantly in proliferating cells. Further analysis indicated that, in the quiescent cells, p18(INK4c) is found in increasing association with CDK6, whereas p27(Kip1) associates predominantly with CDK2. In MEL28 cells, CDK2 activity is completely abrogated in the inner regions of the spheroid, whereas in EMT6 cells, CDK2 activity decreases in accordance with a decrease in expression. We also observed a decrease in all cell cycle regulatory proteins in the innermost spheroid fraction, including CDKs, CKIs, and cyclins. Induction of CKIs from separate families, as well as their association with distinct target CDKs, suggests that there may be multiple checkpoints activated to ensure cell cycle arrest in non-growth-conducive environments. Furthermore, because very similar observations were made in both a human melanoma cell line and a mouse mammary carcinoma cell line, our results indicate that these checkpoints, as well as the signal transduction pathways that activate them, are highly conserved.